http://2007.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Kdikaiou2007.igem.org - User contributions [en]2024-03-29T08:11:29ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/ETHZETHZ2007-10-26T19:19:35Z<p>Kdikaiou: 1st page rearrangement.</p>
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<p align="center"><font size="6"><b>ETH Zurich - educatETH <i>E.coli</i> System</b></font></p><br />
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=Introduction=<br />
<blockquote>"All <i>E.coli</i> 's are equal, but some <i>E.coli</i> 's are more equal than others..." ''(freely adapted from "Animal Farm" by George Orwell)''</blockquote> <br />
<br />
... this is what George Orwell would have written, were he a synthetic biologist. In the <i>E.coli</i> colonies on petri dishes, all bacteria are equal; except for some special ones. Our project is about designing such special <i>E.coli</i> that are "more equal" than the rest: they have the ability to be trained in order to memorize and recognize their environment in the future. Their story will be presented through this wiki ...<br />
<br />
==Motivation==<br />
<br />
[[Image:ethz_main_pic.jpg|right|thumb|<b>Fig. 1</b>: Artist's approach to the different stages of the development. We started by modeling and simulating the system. We continued by specifying the DNA strands for its implementation. Finally in the end, our system should report with different fluorescent proteins (image edited)|600px]]<br />
<br />
Our combined team of biologists and engineers is coping with the problem of implementing memory capabilities in bacterial colonies. First, ''E.coli''are able to respond differently (with distinct fluorescent proteins) to two different inputs (we used chemicals). Second, they remember which input was presented to them. Finally, when confronted with a new input, they are able to recognize whether it is the one that they were trained with or not. <br />
In other words, in this project '''we are educating the <i>E. coli</i>'''!<br />
<br />
===Multipurpose Cell Lines===<br />
<br />
Our system has the ability to behave in different ways according to an internal toggle inside it switching states based on the chemical substances that the system is exposed to. The toggle states could generally be used to trigger events such as enzyme synthesis, transcriptional regulation, virion production, or even cell death. Therefore, one may view the bacterial cell line containing this system as a multipurpose cell line. By adding a certain chemical to a cell line, the latter may be trained to exhibit a desired behavior, and then it is not necessary any more to construct two independent cell lines. <br />
<br />
This means that one applies an “input engineering” instead of a “DNA engineering” approach. If one extends this idea to several inducible toggle switches being harbored in the same cell line, the number of possible phenotypes increases to 2<sup>n</sup>, where n equals the number of toggle switches. For example, if one would have 5 such toggle switches inside a cell line, 32 different behavior patterns would be possible. <br />
<br />
For the purpose of creating a toggle switch that is activated in a specific phase only and not always (a multi-inducible toggle switch), as is required for stable biological automatons, we introduced the concept of [https://2007.igem.org/ETHZ/Biology/parts#double_promoters double promoters] to the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts], which can be helpful for engineering systems which exhibit a desired behavior only at specific times. As a concept, double promoters are expandable to multiple promoter sites - this, however, needs further investigation. <br />
<br />
===Link to Epigenetics===<br />
Epigenetics refers to features like chromatin or DNA modifications that do not involve changes in the underlying DNA sequence and are stable over many cell divisions [1],[2]. If one has a closer look at our proposed system, one can also view it as a model-system for epigenetics: Although the DNA sequence itself stays the same, two different subpopulations of cells with different phenotypes can develop from it. Put simply, depending in which state (subpopulation) the toggle switch is, the cells will produce different fluorescent proteins upon addition of two different inducer molecules. Therefore, the epigenetic feature here is the binding of specific repressor proteins whose production is dependent on the toggle switch state.<br />
<br />
===Intelligent Biosensors and Self-Adaptation===<br />
<br />
The system is capable of sensing different chemicals and producing different fluorescent proteins. Since the cells can be trained to produce one of several specific fluorescent protein types when a certain chemical is present, one can also view those cells as intelligent biosensors which recognize chemical substances according to a training phase. The intelligent biosensors are not limited to detect chemicals; temperature, pH, light, pressure could be detected with an appropriate system as well. Such an application could be especially of interest when the environment to be probed is harmful for humans, for example due to high toxicity.<br />
<br />
==Team Members==<br />
[[Image:ETHZ_Group_photo_6.png|right|thumb|The ETH Zurich iGEM2007 Team|250px]]<br />
<br />
The ETH Zurich team consists of a good mixture between biologists and engineering students. <br />
We are:<br />
<ul> <br />
<li><i>Undergraduate students</i>: <br><br />
[https://2007.igem.org/User:brutsche Martin Brutsche], [https://2007.igem.org/User:kdikaiou Katerina Dikaiou], <br>[https://2007.igem.org/User:Raphael Raphael Guebeli], [https://2007.igem.org/User:hoehnels Sylke Hoehnel], <br><br />
[https://2007.igem.org/Nan_Li Nan Li], <br />
[https://2007.igem.org/User:Stefan Stefan Luzi]<br />
<li><i>Graduate students</i>: <br><br />
[http://christos.bergeles.net Christos Bergeles], <br />
[http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm], <br><br />
[http://www.fussenegger.ethz.ch/people/kemmerc Christian Kemmer], <br />
[https://2007.igem.org/User:JoeKnight Joseph Knight], <br><br />
[http://www.ricomoeckel.de Rico Möckel], <br />
[http://csb.inf.ethz.ch/people/uhr.html Markus Uhr]<br />
<li><i>Project advisors</i>: <br><br />
[http://www.ipe.ethz.ch/laboratories/bpl/people/panke Sven Panke], <br><br />
[http://csb.inf.ethz.ch/people/stelling.html Joerg Stelling]<br />
</ul><br />
<br />
For more information about us, visit our [[ETHZ/Meet_the_team | Meet the Team]] page.<br />
<br />
==Acknowledgments==<br />
The idea for the project as well as its implementation was done solely by the ETH iGEM 2007 team. We would like to thank the people in [http://www.ipe.ethz.ch/laboratories/bpl/index Sven Panke's Lab], especially Andreas Meyer who was always there for us when we had a problem. Additionally, we would like to thank [http://www.facs.ethz.ch Alfredo Franco-Obregón's lab] and Oralea Büchi for the help with the flow cytometry.<br />
<br />
We would also like to acknowledge the financial support by [http://europa.eu EU], the [http://www.ethz.ch ETH Zurich], and [http://www.geneart.com GeneArt]:<br />
<center><br />
{| border="0"<br />
|-<br />
| [http://europa.eu http://www.tik.ee.ethz.ch/~thohm/EU.gif]<br />
| width="40" |<br />
| [http://www.ethz.ch http://www.tik.ee.ethz.ch/~thohm/ethlogo.jpg]<br />
| width="40" |<br />
| [http://www.geneart.com http://www.tik.ee.ethz.ch/~thohm/geneart.gif]<br />
|}<br />
</center><br />
<br />
==Site Map==<br />
<br />
In this wiki, we will present to you a detailed description of the proposed system: starting with the [[ETHZ/Model | modeling of the system]], we describe both, [[ETHZ/Simulation | simulations and theoretical considerations]] of the system, as well as the actual [[ETHZ/Biology | implementation using bio-bricks]] accompanied by our [[ETHZ/Biology/Lab | lab notes]]. Additionally, you will find further [[ETHZ/Meet_the_team | information on the team]], more details about [[ETHZ/Internal | ideas we developed]] before we came up with the system we finally implemented, and some [[ETHZ/Pictures | pictures]] documenting our work.<br />
<br />
The site map of our wiki is the following:<br />
<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="34%"| Modeling Pages <br />
!width="22%"| Biology Pages<br />
!width="22%"| ETHZ Team Pages<br />
!width="22%"| Links<br />
|-<br />
| [[ETHZ/Model | Modeling of the learning system]]<br />
| [[ETHZ/Biology | Biological implementation]]<br />
| [[ETHZ/Meet_the_team | Team page]]<br />
| [https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]<br />
|-<br />
| [[ETHZ/FlipFlop | Representation using flip-flops]]<br />
| [[ETHZ/Biology/parts | Biobricks/parts]]<br />
| [[ETHZ/Pictures | Pictures]]<br />
| [https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]<br />
|-<br />
| [[ETHZ/FSM | Representation using finite state machines]]<br />
| [[ETHZ/Biology/Lab | Lab notes]]<br />
| [[ETHZ/Internal | Brainstorming sessions]]<br />
| <br />
|-<br />
| [[ETHZ/Simulation | Model simulations and theoretical considerations]]<br />
| <br />
| <br />
| <br />
|-<br />
| [[ETHZ/Parameters | Parameters used in our simulations]]<br />
| <br />
| <br />
| <br />
|-<br />
|}<br />
<br />
== References ==<br />
<br />
[http://www.nature.com/nature/journal/v447/n7143/abs/nature05913.html;jsessionid=62903C604764B175945C03DB8639ECBD &#91;1&#93; Bird A] <i>"Perceptions of epigenetics"</i>, Nature 447:396-398, 2007 <br /><br />
[http://linkinghub.elsevier.com/retrieve/pii/S096098220701007X &#91;2&#93; Ptashne M] <i>"On the use of the word ‘epigenetic’"</i>, Current Biology 17(7):R233-R236, 2007 <br /><br />
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= Parameters for the educatETH <i>E.coli</i> system =<br />
<br />
<p><br />
In order to ensure that our simulation results match the biology as close as possible, we tried to find good estimates for the biological system parameters. To this end, we performed an extensive literature review. However, not all parameters were found in the literature, especially those which do not refer to vastly used proteins. Whenever this was the case, logical estimates were provided by our biologists based on empirical values.<br />
</p><br><br />
<br />
== Model Parameters ==<br />
<br />
=== General parameters ===<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| c<sub>1</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of constitutive promoter (per gene)<br />
| promoter no. J23105; Estimate<br />
|-<br />
| c<sub>2</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of LuxR-activated promoter (per gene)<br />
| Estimate<br />
|-<br />
| l<sup>hi</sup><br />
| 25<br />
| high-copy plasmid number<br />
| Estimate<br />
|-<br />
| l<sup>lo</sup><br />
| 5<br />
| low-copy plasmid number<br />
| Estimate<br />
|-<br />
| a<br />
| 1%<br />
| basic production levels<br />
| Estimate<br />
|-<br />
|}<br />
<br />
=== Degradation constants ===<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| d<sub>LacI</sub><br />
| 2.31e-3 [1/s]<br />
| degradation of LacI<br />
| Ref. [10]<br />
|-<br />
| d<sub>TetR</sub><br />
| <br />
*1e-5 [1/s]<br />
*2.31e-3 [1/s]<br />
| degradation of TetR<br />
| <br />
*Ref. [9]<br />
*Ref. [10]<br />
|-<br />
| d<sub>LuxR</sub><br />
| 1e-3 - 1e-4 [1/s]<br />
| degradation of LuxR<br />
| Ref: [6]<br />
|-<br />
| d<sub>CI</sub><br />
| 7e-4 [1/s]<br />
| degradation of CI<br />
| Ref. [7]<br />
|-<br />
| d<sub>P22CII</sub><br />
| <br />
| degradation of P22CII<br />
| <br />
|-<br />
| d<sub>YFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of YFP<br />
| suppl. mat. to Ref. [8] corresponding to a half life of 110min<br />
|-<br />
| d<sub>GFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of GFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>RFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of RFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>CFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of CFP<br />
| in analogy to YFP<br />
|-<br />
|}<br />
<br />
=== Dissociation constants ===<br />
<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| K<sub>LacI</sub><br />
| <br />
* 0.1 - 1 [pM]<br />
* 800 [nM]<br />
| LacI repressor dissociation constant<br />
| <br />
* Ref. [2]<br />
* Ref. [12]<br />
|-<br />
| K<sub>IPTG</sub><br />
| 1.3 [&#181;M]<br />
| IPTG-LacI repressor dissociation constant<br />
| Ref. [2]<br />
|-<br />
| K<sub>TetR</sub><br />
| 179 [pM]<br />
| TetR repressor dissociation constant<br />
| Ref. [1]<br />
|-<br />
| K<sub>ATC</sub><br />
| 893 [pM]<br />
| ATC-TetR repressor dissociation constant<br />
| Ref. [1]<br />
|-<br />
| K<sub>LuxR</sub><br />
| 55 - 520 [nM]<br />
| LuxR activator dissociation constant<br />
| Ref: [6]<br />
|-<br />
| K<sub>AHL</sub><br />
| 0.09 - 1 [&#181;M]<br />
| AHL-LuxR activator dissociation constant<br />
| Ref: [6]<br />
|-<br />
| K<sub>CI</sub><br />
|<br />
*8 [pM]<br />
*50 [nM]<br />
| CI repressor dissociation constant<br />
|<br />
*Ref. [12]<br />
*starting with values of Ref. [6] and using Ref. [3]<br />
|-<br />
| K<sub>P22CII</sub><br />
| 0.577 [&#181;M]<br />
| P22CII repressor dissociation constant<br />
| Ref. [11]<br />
|-<br />
|}<br />
<br />
=== Hill cooperativity ===<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| n<sub>LacI</sub><br />
| <br />
* 1<br />
* 2<br />
| LacI repressor Hill cooperativity<br />
| <br />
* Ref. [5]<br />
* Ref. [12]<br />
|-<br />
| n<sub>IPTG</sub><br />
| 2<br />
| IPTG-LacI repressor Hill cooperativity<br />
| Ref. [5]<br />
|-<br />
| n<sub>TetR</sub><br />
| 3<br />
| TetR repressor Hill cooperativity<br />
| Ref. [3]<br />
|-<br />
| n<sub>ATC</sub><br />
| 2 (1.5-2.5)<br />
| ATC-TetR repressor Hill cooperativity<br />
|Ref. [3]<br />
|-<br />
| n<sub>LuxR</sub><br />
| 2<br />
| LuxR activator Hill cooperativity<br />
| Ref: [6]<br />
|-<br />
| n<sub>AHL</sub><br />
| 1<br />
| AHL-LuxR activator Hill cooperativity<br />
| Ref. [3]<br />
|-<br />
| n<sub>CI</sub><br />
| 2<br />
| CI repressor Hill cooperativity<br />
| Ref. [12]<br />
|-<br />
| n<sub>P22CII</sub><br />
| 4<br />
| P22CII repressor Hill cooperativity<br />
| Ref. [11]<br />
|-<br />
|}<br />
<br />
<br><br />
<br />
== References ==<br />
<p><br />
[http://www.pnas.org/cgi/content/abstract/104/8/2643 &#91;1&#93; Weber W et al.] <i>"A synthetic time-delay circuit in mammalian cells and mice"</i>, P Natl Acad Sci USA 104(8):2643-2648, 2007<br /><br />
[http://www.pnas.org/cgi/content/full/100/13/7702?ck=nck &#91;2&#93; Setty Y et al.] <i>"Detailed map of a cis-regulatory input function"</i>, P Natl Acad Sci USA 100(13):7702-7707, 2003<br /><br />
[http://ieeexplore.ieee.org/iel5/9711/30654/01416417.pdf &#91;3&#93; Braun D et al.] <i>"Parameter Estimation for Two Synthetic Gene Networks: A Case Study"</i>, ICASSP 5:769-772, 2005<br /><br />
[http://www.nature.com/nature/journal/v435/n7038/suppinfo/nature03508.html &#91;4&#93; Fung E et al.] <i>"A synthetic gene--metabolic oscillator"</i>, Nature 435:118-122, 2005 (supplementary material)<br /><br />
[http://dx.doi.org/10.1016/j.jbiotec.2005.08.030 &#91;5&#93; Iadevaia S and Mantzais NV] <i>"Genetic network driven control of PHBV copolymer composition"</i>, J Biotechnol 122(1):99-121, 2006<br /><br />
[http://dx.doi.org/10.1016/j.biosystems.2005.04.006 &#91;6&#93; Goryachev AB et al.] <i>"Systems analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constants"</i>, Biosystems 83(2-3):178-187, 2004<br /><br />
[http://www.genetics.org/cgi/content/abstract/149/4/1633 &#91;7&#93; Arkin A et al.] <i>"Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-Infected Escherichia coli cells"</i>, Genetics 149: 1633-1648, 1998<br /><br />
[http://download.cell.com/supplementarydata/cell/107/6/739/DC1/index.htm &#91;8&#93; Colman-Lerner A et al.] <i>"Yeast Cbk1 and Mob2 Activate Daughter-Specific Genetic Programs to Induce Asymmetric Cell Fates"</i>, Cell 107(6): 739-750, 2001 (supplementary material)<br /><br />
[http://www.nature.com/nature/journal/v405/n6786/abs/405590a0.html &#91;9&#93; Becskei A and Serrano L] <i>"Engineering stability in gene networks by autoregulation"</i>, Nature 405: 590-593, 2000<br /><br />
[http://www.biophysj.org/cgi/content/full/89/6/3873?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1&FIRSTINDEX=0&volume=89&firstpage=3873&resourcetype=HWCIT &#91;10&#93; Tuttle et al.] <i>"Model-Driven Designs of an Oscillating Gene Network"</i>, Biophys J 89(6):3873-3883, 2005<br /><br />
[http://www.pnas.org/cgi/reprint/99/2/679 &#91;11&#93; McMillen LM et al.] <i>"Synchronizing genetic relaxation oscillators by intercell signaling"</i>, P Natl Acad Sci USA 99(2):679-684, 2002<br /><br />
[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html &#91;12&#93; Basu S et al.] <i>"A synthetic multicellular system for programmed pattern formation"</i>, Nature 434:1130-1134, 2005<br /></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-25T20:15:57Z<p>Kdikaiou: </p>
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__NOTOC__<br />
<br />
= Introduction =<br />
<br />
On this page, you can find an analysis of the function of our system, its biological design, and a list of the parts that make up the system. Under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered.<br />
<br />
EducatETH <i>E.coli</i> is a system which can distinguish between [http://openwetware.org/wiki/ATc anhydrotetracycline (aTc)] and [http://openwetware.org/wiki/IPTG Isopropyl-beta-D-thiogalactopyranoside (IPTG)] based on a previous learning phase conducted with the same chemicals and the help of [http://partsregistry.org/Acyl-HSLs acylhomoserine lactone (AHL)]. It is composed of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (LacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists an extended version of the original toggle switch found in [1]. That is, a multi-inducible toggle switch. The main difference is reflected in the use of double promoters, so that the toggle switch only changes its state when both, one of the two chemicals (aTc/IPTG), and AHL are present. As AHL is only present during the learning phase, the toggle maintains its state during testing/recognition, and thus can “memorize”. AHL can therefore be seen as a training- or learning substance. In the reporting subsystem, four reporters ([http://partsregistry.org/Featured_Parts:Fluorescent_proteins fluorescent proteins]) allow supervision of (1.) the chemical the system was trained with and (2.) if the system recognizes the chemical it is being exposed to in the recognition phase as one it has been previously trained with or not.<br />
<br />
== The Complete System ==<br />
<br />
<p>[[Image:Biol_system_stand24.10.png|thumb|left|350px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] The biological design of educatETH <i>E.coli</i> is presented in Fig. 1 and below, we clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modeled? Then visit the [[ETHZ/Model| System Modeling]]!)</p><br />
<br />
==== Constitutive Subsystem ====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). For now, we will consider that AHL is absent and therefore LuxR cannot activate transcription. The TetR and LacI parts behave similarly: more specifically, the TetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus p22cII is produced). Correspondingly, cI is produced when IPTG is present.</p><br />
<br />
==== Learning Subsystem ====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (whose production has in turn been induced by aTc). The LuxR-AHL complex induces cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). In analogy to the upper part, the LuxR-AHL complex induces production of p22cII and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI in the system, which in turn was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
==== Reporting Subsystem ====<br />
<br />
<p>There are four reporters in the system. CFP (more precisely: enhanced CFP, that is ECFP) and YFP (more precisely: enhanced YFP, that is EYFP) are active during the learning phase of the system and show which chemical the system is exposed to during learning, whereas all four reporters (the latter and GFP and RFP) are active during the recognition phase and show if the system is exposed to the same chemical as in learning or not. <br />
More specifically, the YFP production is regulated with help of two operator sites controlled by cI and aTc (TetR inhibitor). cI inhibits the YFP production and aTc induces it. Therefore, YFP is synthesized when the system is exposed to only aTc and cI is not produced within the system (i.e. the system has not been previously exposed to IPTG). The production of the other fluorescent proteins is regulated in a similar manner. Overall, the production of the fluorescent proteins is regulated as follows:<br />
*YFP gets produced when the system is exposed to only aTc and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*CFP gets produced when the system is exposed to only IPTG and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).<br />
*GFP gets produced when the system is exposed to only IPTG and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*RFP gets produced when the system is exposed to only aTc and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).</p><br />
<br />
This behaviour is visualized in Fig. 2.<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|350px|<b>Fig. 2</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== System Phases ==<br />
<br />
<p>The system operation is divided into three main phases: a learning phase, a memory phase and a recognition phase. During the learning phase, the system is first exposed to one of the two chemicals it is designed to detect (aTc or IPTG). During the memory phase, the specific chemical (aTc or IPTG) is removed and AHL is added to activate the systems internal toggle switch. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was detected) or CFP (if IPTG was detected). During the recognition phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. Lets compare the systems toggle switch state with the effect of the newly introduced chemical: the system shows a different response if it has previously been exposed to this certain chemical and reports with the same XFP as in the learning phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc). The following table represents all possible paths that may be taken by the system during all phases of operation according to external stimuli: </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
!width="44" style="background:#446084; color:white"| aTc<br />
!width="44" style="background:#446084; color:white"| IPTG<br />
!width="44" style="background:#446084; color:white"| AHL<br />
!width="44" style="background:#446084; color:white"| p22cII<br />
!width="44" style="background:#446084; color:white"| cI<br />
! style="background:#446084; color:white"| Reporting <br />
|- <br />
|colspan="7" style="background:#96c9cf;" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| non<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP (fading)<br>finally no color<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP (fading)<br>finally no color<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
== System Parts ==<br />
<br />
<p>educatETH <i>E.coli</i> was implemented with 11 basic parts designed by the ETH Zurich team. [https://2007.igem.org/wiki/index.php?title=ETHZ/Biology/parts The list of all the parts, plasmids and strains used] is available. Because the part information is retrieved from the Registry, the page needs some time to load. <br>(Are you interested in this information because you want to implement educatETH <i>E.coli</i> in your lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!)</p><br />
<br />
== References ==<br />
<br />
[http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html &#91;1&#93; Gardner TS, Cantor CR and Collins JJ] <i>"Construction of a genetic toggle switch in Escherichia coli"</i>, Nature 403:339–342, 2000<br /></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/User:KdikaiouUser:Kdikaiou2007-10-24T20:28:02Z<p>Kdikaiou: </p>
<hr />
<div>[[Image:Katerina_Dikaiou.jpg|left|90px]]Hello there! I am Katerina - one of the engineers in the group. I hold a Diploma in in Electrical and Computer Engineering from the Aristotle University of Thessaloniki, Greece, and since October 2006 I have been a Master student of Biomedical Engineering at the ETH Zurich. I am a student member of the IEEE (Engineering in Medicine and Biology Society, Signal Processing Society) and a member of the Technical Chamber of Greece. <br />
<br />
I wanted to take part in iGEM 2007 because I thought it would be a great opportunity to improve my biology knowledge and work with a student team on a project we chose and implemented from the beginning to the end!<br />
<br />
Apart from studies, I enjoy taking very long walks, listening to music (currently a lot of Cocteau Twins), traveling and learning new languages (currently Spanish, but don't ask me grammar questions just yet :)) I look forward to meeting you all in Boston!</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-24T20:00:30Z<p>Kdikaiou: </p>
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<br />
= Introduction =<br />
<br />
On this page, you can find an analysis of the function of our system, its biological design, and a list of the parts that make up the system. Under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered.<br />
<br />
EducatETH <i>E.coli</i> is a system which can distinguish between [http://openwetware.org/wiki/ATc anhydrotetracycline (aTc)] and [http://openwetware.org/wiki/IPTG Isopropyl-beta-D-thiogalactopyranoside (IPTG)] based on a previous learning phase conducted with the same chemicals and the help of [http://partsregistry.org/Acyl-HSLs AHL]. It is composed of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (LacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists an extended version of the original toggle switch found in [1]. That is, a multi-inducible toggle switch. The main difference is reflected in the use of double promoters, so that the toggle switch only changes its state when both, one of the two chemicals (aTc/IPTG), and AHL are present. As AHL is only present during the learning phase, the toggle maintains its state during testing/recognition, and thus can “memorize”. AHL can therefore be seen as a training- or learning substance. In the reporting subsystem, four reporters ([http://partsregistry.org/Featured_Parts:Fluorescent_proteins fluorescent proteins]) allow supervision of (1.) the chemical the system was trained with and (2.) if the system recognizes the chemical it is being exposed to in the recognition phase as one it has been previously trained with or not.<br />
<br />
== The Complete System ==<br />
<br />
<p>[[Image:Biol_system_stand24.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] The biological design of educatETH <i>E.coli</i> is presented in Fig. 1 and below, we clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modeled? Then visit the [[ETHZ/Model| System Modeling]]!)</p><br />
<br />
==== Constitutive Subsystem ====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). For now, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and LacI parts behave similarly: more specifically, the TetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus p22cII is produced). Correspondingly, cI is produced when IPTG is present.</p><br />
<br />
==== Learning Subsystem ====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has in turn been induced by aTc). The LuxR-AHL complex induces cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). In analogy to the upper part, the LuxR-AHL complex induces production of p22cII and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI in the system, which in turn was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
==== Reporting Subsystem ====<br />
<br />
<p>There are four reporters in the system. CFP (more precisely: enhanced CFP, that is ECFP) and YFP (more precisely: enhanced YFP, that is EYFP) are active during the learning phase of the system and show which chemical the system is exposed to during learning, whereas all four reporters (the latter and GFP and RFP) are active during the recognition phase and show if the system is exposed to the same chemical as in learning or not. <br />
More specifically, the YFP production is regulated with help of two operator sites controlled by cI and aTc (TetR inhibitor). cI inhibits the YFP production and aTc induces it. Therefore, YFP is synthesized when the system is exposed to only aTc and cI is not produced within the system (i.e. the system has not been previously exposed to IPTG). The production of the other fluorescent proteins is regulated in a similar manner. Overall, the production of the fluorescent proteins is regulated as follows:<br />
*YFP gets produced when the system is exposed to only aTc and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*CFP gets produced when the system is exposed to only IPTG and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).<br />
*GFP gets produced when the system is exposed to only IPTG and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*RFP gets produced when the system is exposed to only aTc and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).</p><br />
<br />
This behaviour is visualized in Fig. 1.<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== System Phases ==<br />
<br />
<p>The system operation is divided into three main phases: a learning phase, a memory phase and a recognition phase. During the learning phase, the system is first exposed to one of the two chemicals it is designed to detect (aTc or IPTG). During the memory phase, the specific chemical (aTc or IPTG) is removed and AHL is added to activate the systems internal toggle switch. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was detected) or CFP (if IPTG was detected). During the recognition phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. Lets compare the systems toggle switch state with the effect of the newly introduced chemical: the system shows a different response if it has previously been exposed to this certain chemical and reports with the same XFP as in the learning phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc). The following table represents all possible paths that may be taken by the system during all phases of operation according to external stimuli: </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
!width="44" style="background:#446084; color:white"| aTc<br />
!width="44" style="background:#446084; color:white"| IPTG<br />
!width="44" style="background:#446084; color:white"| AHL<br />
!width="44" style="background:#446084; color:white"| p22cII<br />
!width="44" style="background:#446084; color:white"| cI<br />
! style="background:#446084; color:white"| Reporting <br />
|- <br />
|colspan="7" style="background:#96c9cf;" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| non<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP (fading)<br>finally no color<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP (fading)<br>finally no color<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
== System Parts ==<br />
<br />
<p>educatETH <i>E.coli</i> was implemented with 11 parts designed by the ETH Zurich team. [hhttps://2007.igem.org/wiki/index.php?title=ETHZ/Biology&action=submit The list of all the parts used] is available. Because the part information is retrieved from the Registry, the page needs some time to load. <br>(Are you interested in this information because you want to implement educatETH <i>E.coli</i> in your lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!)</p><br />
<br />
== References ==<br />
<p><br />
[http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html &#91;1&#93; Gardner TS, Cantor CR and Collins JJ] <i>"Construction of a genetic toggle switch in Escherichia coli"</i>, Nature 403:339–342, 2000<br /><br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:blue;>Katerina's note: Put Sylke's new scheme on this page. Note: the scheme is ready, it just needs to be uploaded. DONE</span><br />
<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:Biol_system_stand24.10.pngFile:Biol system stand24.10.png2007-10-24T19:58:51Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-24T19:58:31Z<p>Kdikaiou: New image</p>
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__NOTOC__<br />
<br />
= Introduction =<br />
<br />
On this page, you can find an analysis of the function of our system, its biological design, and a list of the parts that make up the system. Under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered.<br />
<br />
EducatETH <i>E.coli</i> is a system which can distinguish between [http://openwetware.org/wiki/ATc anhydrotetracycline (aTc)] and [http://openwetware.org/wiki/IPTG Isopropyl-beta-D-thiogalactopyranoside (IPTG)] based on a previous learning phase conducted with the same chemicals and the help of [http://partsregistry.org/Acyl-HSLs AHL]. It is composed of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (LacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists an extended version of the original toggle switch found in [1]. That is, a multi-inducible toggle switch. The main difference is reflected in the use of double promoters, so that the toggle switch only changes its state when both, one of the two chemicals (aTc/IPTG), and AHL are present. As AHL is only present during the learning phase, the toggle maintains its state during testing/recognition, and thus can “memorize”. AHL can therefore be seen as a training- or learning substance. In the reporting subsystem, four reporters ([http://partsregistry.org/Featured_Parts:Fluorescent_proteins fluorescent proteins]) allow supervision of (1.) the chemical the system was trained with and (2.) if the system recognizes the chemical it is being exposed to in the recognition phase as one it has been previously trained with or not.<br />
<br />
== The Complete System ==<br />
<br />
<p>[[Image:Biol_system_stand24.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] The biological design of educatETH <i>E.coli</i> is presented in Fig. 1 and below, we clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modeled? Then visit the [[ETHZ/Model| System Modeling]]!)</p><br />
<br />
==== Constitutive Subsystem ====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). For now, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and LacI parts behave similarly: more specifically, the TetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus p22cII is produced). Correspondingly, cI is produced when IPTG is present.</p><br />
<br />
==== Learning Subsystem ====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has in turn been induced by aTc). The LuxR-AHL complex induces cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). In analogy to the upper part, the LuxR-AHL complex induces production of p22cII and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI in the system, which in turn was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
==== Reporting Subsystem ====<br />
<br />
<p>There are four reporters in the system. CFP (more precisely: enhanced CFP, that is ECFP) and YFP (more precisely: enhanced YFP, that is EYFP) are active during the learning phase of the system and show which chemical the system is exposed to during learning, whereas all four reporters (the latter and GFP and RFP) are active during the recognition phase and show if the system is exposed to the same chemical as in learning or not. <br />
More specifically, the YFP production is regulated with help of two operator sites controlled by cI and aTc (TetR inhibitor). cI inhibits the YFP production and aTc induces it. Therefore, YFP is synthesized when the system is exposed to only aTc and cI is not produced within the system (i.e. the system has not been previously exposed to IPTG). The production of the other fluorescent proteins is regulated in a similar manner. Overall, the production of the fluorescent proteins is regulated as follows:<br />
*YFP gets produced when the system is exposed to only aTc and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*CFP gets produced when the system is exposed to only IPTG and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).<br />
*GFP gets produced when the system is exposed to only IPTG and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*RFP gets produced when the system is exposed to only aTc and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).</p><br />
<br />
This behaviour is visualized in Fig. 1.<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== System Phases ==<br />
<br />
<p>The system operation is divided into three main phases: a learning phase, a memory phase and a recognition phase. During the learning phase, the system is first exposed to one of the two chemicals it is designed to detect (aTc or IPTG). During the memory phase, the specific chemical (aTc or IPTG) is removed and AHL is added to activate the systems internal toggle switch. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was detected) or CFP (if IPTG was detected). During the recognition phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. Lets compare the systems toggle switch state with the effect of the newly introduced chemical: the system shows a different response if it has previously been exposed to this certain chemical and reports with the same XFP as in the learning phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc). The following table represents all possible paths that may be taken by the system during all phases of operation according to external stimuli: </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
!width="44" style="background:#446084; color:white"| aTc<br />
!width="44" style="background:#446084; color:white"| IPTG<br />
!width="44" style="background:#446084; color:white"| AHL<br />
!width="44" style="background:#446084; color:white"| p22cII<br />
!width="44" style="background:#446084; color:white"| cI<br />
! style="background:#446084; color:white"| Reporting <br />
|- <br />
|colspan="7" style="background:#96c9cf;" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| non<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP (fading)<br>finally no color<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP (fading)<br>finally no color<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
== System Parts ==<br />
<br />
<p>educatETH <i>E.coli</i> was implemented with 11 parts designed by the ETH Zurich team. [hhttps://2007.igem.org/wiki/index.php?title=ETHZ/Biology&action=submit The list of all the parts used] is available. Because the part information is retrieved from the Registry, the page needs some time to load. <br>(Are you interested in this information because you want to implement educatETH <i>E.coli</i> in your lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!)</p><br />
<br />
== References ==<br />
<p><br />
[http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html &#91;1&#93; Gardner TS, Cantor CR and Collins JJ] <i>"Construction of a genetic toggle switch in Escherichia coli"</i>, Nature 403:339–342, 2000<br /><br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:red;>Katerina's note: Put Sylke's new scheme on this page. Note: the scheme is ready, it just needs to be uploaded.</span><br />
<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/ParametersETHZ/Parameters2007-10-24T19:18:29Z<p>Kdikaiou: Put drop-down menu on the top.</p>
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__NOTOC__<br />
<br />
<br />
= Parameters for the EducatETH <i>E. coli</i> system =<br />
<br />
<p><br />
In order to provide as realistic simulation results as possible, and to find good estimates for the simulation parameters, we performed an intensive literature review. However, not all parameters could be found in the literature. Furthermore, one has to take into account that biological parameters cannot be estimated to a very high precision.<br />
</p><br><br />
<br />
== Model Parameters ==<br />
<br />
=== General parameters ===<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| c<sub>1</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of constitutive promoter (per gene)<br />
| promoter no. J23105; Estimate<br />
|-<br />
| c<sub>2</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of LuxR-activated promoter (per gene)<br />
| Estimate<br />
|-<br />
| l<sup>hi</sup><br />
| 25<br />
| high-copy plasmid number<br />
| Estimate<br />
|-<br />
| l<sup>lo</sup><br />
| 5<br />
| low-copy plasmid number<br />
| Estimate<br />
|-<br />
| a<br />
| 1%<br />
| basic production levels<br />
| Estimate<br />
|-<br />
|}<br />
<br />
=== Degradation constants ===<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| d<sub>LacI</sub><br />
| 2.31e-3 [1/s]<br />
| degradation of LacI<br />
| Ref. [10]<br />
|-<br />
| d<sub>TetR</sub><br />
| <br />
*1e-5 [1/s]<br />
*2.31e-3 [1/s]<br />
| degradation of TetR<br />
| <br />
*Ref. [9]<br />
*Ref. [10]<br />
|-<br />
| d<sub>LuxR</sub><br />
| 1e-3 - 1e-4 [1/s]<br />
| degradation of LuxR<br />
| Ref: [6]<br />
|-<br />
| d<sub>CI</sub><br />
| 7e-4 [1/s]<br />
| degradation of CI<br />
| Ref. [7]<br />
|-<br />
| d<sub>P22CII</sub><br />
| <br />
| degradation of P22CII<br />
| <br />
|-<br />
| d<sub>YFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of YFP<br />
| suppl. mat. to Ref. [8] corresponding to a half life of 110min<br />
|-<br />
| d<sub>GFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of GFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>RFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of RFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>CFP</sub><br />
| 6.3e-3 [1/min]<br />
| degradation of CFP<br />
| in analogy to YFP<br />
|-<br />
|}<br />
<br />
=== Dissociation constants ===<br />
<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| K<sub>LacI</sub><br />
| <br />
* 0.1 - 1 [pM]<br />
* 800 [nM]<br />
| LacI repressor dissociation constant<br />
| <br />
* Ref. [2]<br />
* Ref. [12]<br />
|-<br />
| K<sub>IPTG</sub><br />
| 1.3 [&#181;M]<br />
| IPTG-LacI repressor dissociation constant<br />
| Ref. [2]<br />
|-<br />
| K<sub>TetR</sub><br />
| 179 [pM]<br />
| TetR repressor dissociation constant<br />
| Ref. [1]<br />
|-<br />
| K<sub>ATC</sub><br />
| 893 [pM]<br />
| ATC-TetR repressor dissociation constant<br />
| Ref. [1]<br />
|-<br />
| K<sub>LuxR</sub><br />
| 55 - 520 [nM]<br />
| LuxR activator dissociation constant<br />
| Ref: [6]<br />
|-<br />
| K<sub>AHL</sub><br />
| 0.09 - 1 [&#181;M]<br />
| AHL-LuxR activator dissociation constant<br />
| Ref: [6]<br />
|-<br />
| K<sub>CI</sub><br />
|<br />
*8 [pM]<br />
*50 [nM]<br />
| CI repressor dissociation constant<br />
|<br />
*Ref. [12]<br />
*starting with values of Ref. [6] and using Ref. [3]<br />
|-<br />
| K<sub>P22CII</sub><br />
| 0.577 [&#181;M]<br />
| P22CII repressor dissociation constant<br />
| Ref. [11]. Note that they use a protein CII and we have P22CII. Does that match?<br />
|-<br />
|}<br />
<br />
=== Hill cooperativity ===<br />
{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
!width="10%"| Parameter <br />
!width="10%"| Value<br />
!width="20%"| Description<br />
!width="60%"| Comments<br />
|-<br />
| n<sub>LacI</sub><br />
| <br />
* 1<br />
* 2<br />
| LacI repressor Hill cooperativity<br />
| <br />
* Ref. [5]<br />
* Ref. [12]<br />
|-<br />
| n<sub>IPTG</sub><br />
| 2<br />
| IPTG-LacI repressor Hill cooperativity<br />
| Ref. [5]<br />
|-<br />
| n<sub>TetR</sub><br />
| 3<br />
| TetR repressor Hill cooperativity<br />
| Ref. [3]<br />
|-<br />
| n<sub>ATC</sub><br />
| 2 (1.5-2.5)<br />
| ATC-TetR repressor Hill cooperativity<br />
|Ref. [3]<br />
|-<br />
| n<sub>LuxR</sub><br />
| 2<br />
| LuxR activator Hill cooperativity<br />
| Ref: [6]<br />
|-<br />
| n<sub>AHL</sub><br />
| 1<br />
| AHL-LuxR activator Hill cooperativity<br />
| Ref. [3]<br />
|-<br />
| n<sub>CI</sub><br />
| 2<br />
| CI repressor Hill cooperativity<br />
| Ref. [12]<br />
|-<br />
| n<sub>P22CII</sub><br />
| 4<br />
| P22CII repressor Hill cooperativity<br />
| Ref. [11]. Note that they use a protein CII and we have P22CII. Does that match?<br />
|-<br />
|}<br />
<br />
<br><br />
<br />
== References ==<br />
<p><br />
[http://www.pnas.org/cgi/content/abstract/104/8/2643 &#91;1&#93; Weber W et al.] <i>"A synthetic time-delay circuit in mammalian cells and mice"</i>, P Natl Acad Sci USA 104(8):2643-2648, 2007<br /><br />
[http://www.pnas.org/cgi/content/full/100/13/7702?ck=nck &#91;2&#93; Setty Y et al.] <i>"Detailed map of a cis-regulatory input function"</i>, P Natl Acad Sci USA 100(13):7702-7707, 2003<br /><br />
[http://ieeexplore.ieee.org/iel5/9711/30654/01416417.pdf &#91;3&#93; Braun D et al.] <i>"Parameter Estimation for Two Synthetic Gene Networks: A Case Study"</i>, ICASSP 5:769-772, 2005<br /><br />
[http://www.nature.com/nature/journal/v435/n7038/suppinfo/nature03508.html &#91;4&#93; Fung E et al.] <i>"A synthetic gene--metabolic oscillator"</i>, Nature 435:118-122, 2005 (supplementary material)<br /><br />
[http://dx.doi.org/10.1016/j.jbiotec.2005.08.030 &#91;5&#93; Iadevaia S and Mantzais NV] <i>"Genetic network driven control of PHBV copolymer composition"</i>, J Biotechnol 122(1):99-121, 2006<br /><br />
[http://dx.doi.org/10.1016/j.biosystems.2005.04.006 &#91;6&#93; Goryachev AB et al.] <i>"Systems analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constants"</i>, Biosystems 83(2-3):178-187, 2004<br /><br />
[http://www.genetics.org/cgi/content/abstract/149/4/1633 &#91;7&#93; Arkin A et al.] <i>"Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-Infected Escherichia coli cells"</i>, Genetics 149: 1633-1648, 1998<br /><br />
[http://download.cell.com/supplementarydata/cell/107/6/739/DC1/index.htm &#91;8&#93; Colman-Lerner A et al.] <i>"Yeast Cbk1 and Mob2 Activate Daughter-Specific Genetic Programs to Induce Asymmetric Cell Fates"</i>, Cell 107(6): 739-750, 2001 (supplementary material)<br /><br />
[http://www.nature.com/nature/journal/v405/n6786/abs/405590a0.html &#91;9&#93; Becskei A and Serrano L] <i>"Engineering stability in gene networks by autoregulation"</i>, Nature 405: 590-593, 2000<br /><br />
[http://www.biophysj.org/cgi/content/full/89/6/3873?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1&FIRSTINDEX=0&volume=89&firstpage=3873&resourcetype=HWCIT &#91;10&#93; Tuttle et al.] <i>"Model-Driven Designs of an Oscillating Gene Network"</i>, Biophys J 89(6):3873-3883, 2005<br /><br />
[http://www.pnas.org/cgi/reprint/99/2/679 &#91;11&#93; McMillen LM et al.] <i>"Synchronizing genetic relaxation oscillators by intercell signaling"</i>, P Natl Acad Sci USA 99(2):679-684, 2002<br /><br />
[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html &#91;12&#93; Basu S et al.] <i>"A synthetic multicellular system for programmed pattern formation"</i>, Nature 434:1130-1134, 2005<br /></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/LabbookETHZ/Biology/Labbook2007-10-24T19:17:20Z<p>Kdikaiou: </p>
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<br />
== Lab book ==<br />
<br />
==== Week 1 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 06. Aug. 2007 <br />
|<br />
| <br />
* Preparing the Solutions <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Tue, 07. Aug. 2007 <br />
| <br />
|<br />
* Prepare competent cells for all parts<br />
* Transformation of all the parts <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Wed, 08. Aug. 2007 <br />
| <br />
| <br />
* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures) <br />
| Raphael<br>Stefan<br />
|-<br />
| Thu, 09. Aug. 2007 <br />
| <br />
| <br />
* MINIPREP of the grown (10) o/n cultures<br />
* Gelelectrophoresis of the grown cultures (step: 0.8% Agarose) <br />
| Raphael<br>Stefan<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Fri, 10. Aug. 2007 <br />
| <br />
| <br />
* 7 working parts/plasmids (step after "DIGESTS"): <br> (B0034, R0062, R0053, E0434, B0015, R0010, E0422)<br />
* 4 parts/plasmids minipreped: <br> (R0040, R0051, Q04121, C0053)<br />
|<br />
Christos <br> Markus <br> Stefan<br />
|-<br />
| Sat, 11. Aug. 2007 <br />
| <br />
| no labwork <br />
|<br />
|-<br />
| Sun, 12. Aug. 2007 <br />
|<br />
| labwork cancelled<br />
|<br />
|}<br />
<br />
==== Week 2 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 13. Aug. 2007 <br> start at 3 pm <br />
|<br />
* Prepare competent cells<br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053<br />
| <br />
* o/n culture (E.Coli Top10)<br />
* Control Restrictions (didn't work)<br />
| Martin<br> Markus <br> Christos <br> Tim <br><br />
|-<br />
| Tue, 14. Aug. 2007 <br />
| Morning Shift: <br><br />
* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br> <br />
Evening Shift: <br><br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
| Morning Shift: <br><br />
* Prepared competent cells (stored in the -80°C freezer in the basement) <br><br />
Evening Shift: <br><br />
* Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday) <br />
* Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4% <br />
|Morning Shift (9am-1pm?): <br> Markus <br> Tim <br> Evening Shift (5pm-...):<br> Martin <br> Christos<br />
|-<br />
| Wed, 15. Aug. 2007 <br />
| <br />
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434 <br />
|<br />
* Ligation didn't work due to bad quality of enzymes (probably) <br />
| From 12:<br> Martin<br>Markus<br><br><br />
|-<br />
| Thu, 16. Aug. 2007 <br />
| <br />
* Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)<br />
* Transformation of #13 and #14<br />
| <br />
* Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510) <br> One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)<br />
* Transformation of #13 (Q04121) and #14 (C0053) <br> Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)<br />
|<br />
Markus<br>Christos<br>(Martin)<br />
|-<br />
| Fri, 17. Aug. 2007 <br />
|<br />
* o/n of #13 and #14<br />
* Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful<br />
| <br />
* #13 and #14 didn't grow<br />
* # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates<br />
* New white pipette tips prepared (autoclave)<br />
* New bottles of Liquid LB and LB Agar prepared (autoclave)<br />
| Martin<br />
|-<br />
| Sat, 18. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 19. Aug. 2007 <br />
|<br />
|<br />
|<br />
|}<br />
<br />
==== Week 3 ====<br />
<br />
Little rearrangements of the parts. Planning of the sequences to order them.<br />
<br />
<br />
==== Week 4 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 27. Aug. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 28. Aug. 2007 <br />
| <br />
|<br />
| <br />
|-<br />
| Wed, 29. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Thu, 30. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 31. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 01. Sept. 2007 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 and plated them <br />
| Stefan<br />
|-<br />
| Sun, 02. Sept. 2007 <br />
|<br />
* Prepare o/n of pbr322, pcyc177, pck01<br />
| <br />
* o/n of pcyc177, pck01<br />
* the plates of pcyc177 and pck01 are in the fridge<br />
* transformed pbr322 because the culture didn't grow on the plate<br />
| Stefan<br />
|}<br />
<br />
==== Week 5 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 03. Sept. 2007 <br />
|<br />
* Prepare new competent cells<br />
* Miniprep pcyc177 and pcK01<br />
* prepare new o/n culture of pbr322<br />
* Run agarose gel of Minipreped plasmids<br />
| <br />
* New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)<br />
* Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)<br />
* pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday<br />
* new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids<br />
* 2 boxes of blue pipette tips are in the autoclave<br />
* Stefan ran the agarose gel (?) <br />
| Martin<br>Stefan<br />
|-<br />
| Tue, 04. Sept. 2007 <br />
|<br />
* Miniprep pcyc177 and pck01<br />
* cut the prepped plasmids to test if we've got the right ones<br />
* run agarose gel to test the cut and uncut ones <br />
* prepare new o/n of pbr322 (from Andy) <br />
|<br />
* Miniprep of pcyc177 and pck01 (but not yet tested)<br />
* Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep <br />
| Martin<br>Christian<br />
|-<br />
| Wed, 05. Sept. 2007 <br />
| <br />
* Miniprep of pbr322<br />
* Test-Digest of pcyc177 and pck01 and agarose gel...<br />
* Streak out all three plasmids on new plates, so we have them in reserve <br />
|<br />
* New Plate of pbr322.<br />
* Minipreps and Agarose Gels will be done tomorrow <br />
| Martin<br />
|-<br />
| Thu, 06. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01<br />
* Test with agarose gel <br />
| <br />
* Gel of the older plasmids -> plasmid present<br />
| Christian<br />
|-<br />
| Fri, 07. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01 <br />
| <br />
* Plasmids miniprepped<br />
| Martin<br />
|-<br />
| Sat, 08. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 09. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 6 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 10. Sept. 2007 <br />
| <br />
* Miniprep pBR322<br />
* annealing of different MCSs<br />
* Digest of pCK01 with BamHI+AseI<br />
* digest of pACYC177 with BamHI+PstI<br />
* digest of pBR322 with EcoRI+PstI <br />
all digests o/n<br />
| <br />
| <br />
Christian<br />
| <br />
|-<br />
| Tue, 11. Sept. 2007 <br />
|<br />
* Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work<br />
* 1x ligation of MCS inside backbones o/d, Trafo<br />
* 1x ligation of MCS inside backbones o/n<br />
| <br />
* plate all 3 plasmids for new minipreps<br />
|<br />
Christian<br />
|-<br />
| Wed, 12. Sept. 2007 <br />
|<br />
* Trafo of o/n ligations<br />
* o/n cultures of putative clones <br />
|<br />
|<br />
Christian<br />
|-<br />
| Thu, 13. Sept. 2007 <br />
| <br />
* Minipreps of putative clones pCK01-MCS and pBR322-MCS<br />
* control digests of putative clones<br />
* new o/n cultures of the putative clones of o/n ligations<br />
| <br />
| <br />
Christian<br />
|-<br />
| Fri, 14. Sept. 2007 <br />
| <br />
*separation of control digests of putative clones <br />
|<br />
'''*pBR322-MCS (Tet-selection) clone2 positive''' <br />
|<br />
Christian<br />
|-<br />
| Sat, 15. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 16. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 7 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 17. Sept. 2007 <br />
|<br />
* new digest of pACYC177 with BamHI+PstI o/n<br />
* digest of pACYC177, pBR322 AP<br />
* ligation of 177 and 322AP<br />
| <br />
* digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)<br />
* ligation of pBR322 o/n<br />
* 100 ml o/n culture to MAXIprep pacyc177<br />
* Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)<br />
| <br />
Christian <br> Martin, Raphael<br />
|-<br />
| Tue, 18. Sept. 2007 <br />
|<br />
* different control digests of pBR322-MCS (Tet) (see last week)<br />
* separation of pACYC177 digest<br />
* Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)<br />
* Transformation of the ligated pbr322 AP (MCS)<br />
* Prep pacyc177<br />
* Digest prepped pacyc177<br />
|<br />
> no DNA on pACYC177 digest-gel, only degradation smear<br> <br><br />
<br />
* Plates of pbr322 AP grew<br />
* No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)<br />
* Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )<br />
* Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.<br />
* Digest of pacyc177 with PstI o/n (pray that it will work!)<br />
* New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)<br />
* test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests<br />
| <br />
<br />
Christian <br><br />
Martin<br>Raphael<br />
|-<br />
| Wed, 19. Sept. 2007 <br />
| <br />
* o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated<br />
* Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)<br />
* check the digests of pacyc177 (pst) and pck01 (notI)<br />
* design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01 <br />
|<br />
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==== Week 8 ====<br />
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! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
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| Mon, 24. Sept. 2007 <br />
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====Week 9 ====<br />
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! Date <br />
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! Completed <br />
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==== Week 10 ====<br />
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! Date <br />
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|}</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/LabETHZ/Biology/Lab2007-10-24T19:06:05Z<p>Kdikaiou: Put drop-down menu on the top.</p>
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__NOTOC__<br />
<br />
You are <br />
<br />
For all our cloning procedures we used standard protocols according to SAMBROOK and RUSSELL Molecular Cloning: A Laboratory Manual.<br />
<br />
<br />
== Strains ==<br />
<br />
We used the following <i>E. coli </i> strains:<br />
<br />
<br />
[http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29|<b><i>E. coli </i>Top10 (Invitrogen):</b>] <br> <br />
*This strain has a streptomycin resistance <br> <br />
*Genotype: F’ {tetR}, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80 lacZ ΔM15, ΔlacX74, deoR, recA1, araD139 Δ(ara-leu)7679, galU, galK, λ-, rpsL,endA1, nupG<br />
*For further information please [http://openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29| click here]<br />
*<i>References</i>: <br />
**Casdaban, M. and Cohen, S. (1980) J Mol Biol 138:179 PMID 6997493 <br> <br />
**Grant, S.G.N. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 4645-4649 PMID 2162051<br />
<br />
<br />
[http://openwetware.org/wiki/E._coli_genotypes#JM101|<b><i>E. coli </i>JM101:</b>] <br><br />
*We call them <i>Jimmys</i><br />
*This strain is the original blue/white cloning strain<br />
*Genotype: glnV44, thi-1, Δ(lac-proAB), F'[lacIqZΔM15 traD36 proAB+]<br />
*For further information please [http://openwetware.org/wiki/E._coli_genotypes#JM101| click here]<br />
*<i>Reference</i>:<br />
**Messing, J. et al. (1981) Nucleic Acids Res. 9, 309; Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene 33, 103<br />
<br />
== Plasmids ==<br />
<br />
For our system we needed three plasmids with different origins of replication and antibiotic resistances. We decided to take low copy plasmids. We decided to use the following plasmids, which we wanted modify so that they would become compatible to the Biobrick Library multiple cloning site:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|-<br />
! Plasmid !! Resistances !! Copy number !! Origin !! Map <br />
|-<br />
<br />
| [[ETHZ/pbr322| pBR322]] || Ampicillin, Tetracyline || 15-20 [1] || pMB1 || [[Image:Mappbr322.jpg|center|thumb|pBR322 Map|100px]]<br />
|-<br />
<br />
| [[ETHZ/pck01| pCK01]] || Chloramphenicol|| 5-12 [1]|| pSC101 || [[Image:Mappck01.jpg|center|thumb|pCK01 Map|100px]]<br />
|-<br />
<br />
| [[ETHZ/pacyc177| pACYC177]] || Ampicillin, Kanamycin|| 10-12 [1] || p15A || [[Image:Mappacyc177.jpg|center|thumb|pACYC177 Map|100px]]<br />
|-<br />
|}<br />
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In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section [https://2007.igem.org/ETHZ/Biology/Lab#.::_Problems_we_faced_::. Problems we faced] might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Biology | Biology Pespective]]. Finally, photos of our lab experience are accessible under [[ETHZ/Pictures | Pictures!]]<br />
<br />
Todo: decide what happens with lab book ([https://2007.igem.org/ETHZ/Lab_book here])<br />
<br />
==Cloning plan==<br />
<br />
===Parts assignment into plasmids===<br />
<br />
Three plasmids are used for the EducatETH <i>E.coli</i> system parts as follows:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Plasmids and contents'''<br />
|-<br />
! plasmid !! resistance !! copy type!! contents !! comments<br />
|-<br />
<br />
| [[ETHZ/pbr322| pbr322]] || ampicillin || high || 1,2,3 || constitutive subsystem<br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || chloramphenicol|| low || 4,5,8,9 || reporting subsystem<br />
|-<br />
<br />
| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| low || 6,7,10,11 || learning subsystem, reporting subsystem<br />
|-<br />
|}<br />
<br />
It is important to insert parts responsible for the production of fluorescent proteins in low copy plasmids, as they are potentially harmful for the cell. Unfortunately, working with low copy plasmids makes the procedure more demanding in the lab.<br />
<br />
===Linkers===<br />
<br />
Because the plasmids used were not standard plasmids found in the registry, but came from the lab where we work, linkers compatible with the standard BioBrick assembly have to be used in order to work with them. The list of all linkers is the following:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Linkers for plasmids'''<br />
|-<br />
! Linker!! Plasmid<br />
|-<br />
<br />
| [[ETHZ/pbr322-1| pbr322-1]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-2| pbr322-2]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-3| pbr322-3]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-4| pbr322-4]]|| pbr322 <br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || pck01 <br />
|-<br />
| [[ETHZ/pCK01-2| pck01-2]] || pck01 <br />
|-<br />
<br />
| [[ETHZ/pacyc177-1| pacyc177-1]] || pacyc177<br />
|-<br />
| [[ETHZ/pacyc177-2| pacyc177-2]]|| pacyc177<br />
|-<br />
|}<br />
<br />
Note that four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.<br />
<br />
=== Procedure ===<br />
<br />
The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. [[Image:Assembly _process.png|thumb|300px|DNA assembly process ([1]) '''(Fig. 4)''']] Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted ''before'' the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the ''donor'' and the plasmid accepting the new part will be referred to as the ''acceptor''. Composite pars made of parts '''a''' and '''b''' are denoted '''a.b'''.<br />
<br />
==== Plasmid 1 ''(pbr322ap)'' ====<br />
<br />
# Put parts 1,2,3 in pbr322ap plasmids. <br />
# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.<br />
# Merge plasmid containing part '''1''' ''(donor)'' with plasmid containing composite part '''2.3''' ''(acceptor)''. You should get a plasmid containing a '''1.2.3''' composite part.<br />
<br />
==== Plasmid 2 ''(pck01cm)''====<br />
<br />
# Put parts 4,5,8,9 in pck01cm plasmids. <br />
# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.<br />
# Merge plasmid containing part '''8''' ''(donor)'' with plasmid containing part '''9''' ''(acceptor)''. You should get a plasmid containing a '''8.9''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''4.5''' ''(donor)'' with plasmid containing composite part '''8.9''' ''(acceptor)''. You should get a plasmid containing a '''4.5.8.9''' composite part.<br />
<br />
====Plasmid 3 ''(pacyc177km)''====<br />
<br />
# Put parts 6,7,10,11 in pacyc177km plasmids. <br />
# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.<br />
# Merge plasmid containing part '''10''' ''(donor)'' with plasmid containing part '''11''' ''(acceptor)''. You should get a plasmid containing a '''10.11''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''6.7''' ''(donor)'' with plasmid containing composite part '''10.11''' ''(acceptor)''. You should get a plasmid containing a '''6.7.10.11''' composite part.<br />
<br />
== [[ETHZ/Biology/Labbook| Labbook]] ==<br />
<br />
<br />
==References==<br />
<p>[1] [http://www1.qiagen.com/faq/faqview.aspx?faqid=350&SearchText=&FaqCategoryId=0&MenuItemId=0&catalog=1&ProductLineId=1000228 QIAGEN FAQs]</p><br />
<p>[x] [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly Process]</p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/partsETHZ/Biology/parts2007-10-24T19:02:22Z<p>Kdikaiou: Final (?) changes by Katerina [stylistics]</p>
<hr />
<div>= System Parts =<br />
educatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Four of them (4,5 and 8,9) form together two functional system units, but they have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids. All 11 educatETH <i>E.coli</i> are listed in the following table:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1<br />
|width="285"| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo><br />
|}<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Composite System Parts'''<br />
|-<br />
|-<br />
!width="111"| 2+3<br />
|width="285"| lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739012 BBa_I739012]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739012 SpecifiedComponents</partinfo><br />
|-<br />
! 1+2+3<br />
| tetR + lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739013 SpecifiedComponents</partinfo><br />
|-<br />
! 4+5<br />
| P22 cII + EYFP production <br />
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo><br />
|-<br />
! 8+9<br />
| cI + ECFP production <br />
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo><br />
|-<br />
! (4+5)+(8+9)<br />
| (P22 cII + EYFP) + (cI + ECFP) production <br />
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo><br />
|-<br />
! 6+7<br />
| cI + P22 cII production <br />
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]<br />
| learning subsystem<br />
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo><br />
|-<br />
! 10+11<br />
| RFP + GFP production <br />
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo><br />
|-<br />
! (6+7)+(10+11)<br />
| (cI + P22 cII) + (RFP + GFP) production <br />
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]<br />
| learning/reporting subsystem<br />
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
Many of the above mentioned parts contain ''double promoters''. This promoter constructs contain two independent classes of operator sites and can therefore be regulated by two different types of molecules. Double promoters form the basis of the multi-inducible toggle switch and hence the memory of our learning system. This concept could also help future projects in developing devices and systems that need extended regulation. In the following, we introduce a selection of first generation double promoters to the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]: <br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
!width="111"| 1<sup>pro</sup><br />
|width="285"| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
|width="250"| reporting subsystem<br />
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2<sup>pro</sup><br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3<sup>pro</sup><br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4<sup>pro</sup><br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5<sup>pro</sup><br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6<sup>pro</sup><br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
In order to test if the concept of the proposed double promoters is working, simple proof of concept (PoC) parts have been constructed. The PoC promoter, which shows strong similarites to [http://partsregistry.org/Part:BBa_I739102 BBa_I739102], consists of two TetR operator sequences linked to a constitutive promoter. In contrast to the other double promoters , the PoC promoter is only single regulated. The PoC intermediate is part of the PoC composite the concept can be tested with, that is, EYFP production. The p22cII coding region is included to ensure the functionality in multicistronic constructs.<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
!width="111"| 1<sup>poc</sup><br />
|width="285"| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
|width="250"| proof of concept, no part of the system <br />
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2<sup>poc</sup><br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|-<br />
! 3<sup>poc</sup><br />
| PoC composite<br />
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
Although the parts have been synthesized by GENEART and were also shipped in their high-copy plasmids, we desided to change the cloning vectors. This strategy is based on the fact that fluorescent proteins are potentially harmful for the cells. The parts containing DNA sequences coding for these reporter proteins are therefore supposed to be cloned in low-copy number plasmids. In contrast, the constitutive subsystem parts can and should be cloned into a well characterized and working high-copy plasmid. Summarized, the following plasmids have been used:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Plasmids'''<br />
|-<br />
|-<br />
!width="111"| 1<sup>vec</sup><br />
|width="285"| pBR322BB1<br />
| [http://partsregistry.org/Part:BBa_I739201 BBa_I739201]<br />
|width="250"| high-copy cloning vector, ApR<br>constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739201 SpecifiedComponents</partinfo><br />
|-<br />
! 2<sup>vec</sup><br />
| pCK01BB1<br />
| [http://partsregistry.org/Part:BBa_I739202 BBa_I739202]<br />
| low-copy cloning vector, CmR<br>reporting subsystem<br />
| <partinfo>BBa_I739202 SpecifiedComponents</partinfo><br />
|-<br />
! 3<sup>vec</sup><br />
| pCK01BB2<br />
| [http://partsregistry.org/Part:BBa_I739203 BBa_I739203]<br />
| low-copy cloning vector, ApR<br>reporting subsystem<br />
| <partinfo>BBa_I739203 SpecifiedComponents</partinfo><br />
|-<br />
! 4<sup>vec</sup><br />
| pACYC177BB1<br />
| [http://partsregistry.org/Part:BBa_I739204 BBa_I739204]<br />
| low-copy cloning vector, KmR<br>learning and reporting subsystem<br />
| <partinfo>BBa_I739204 SpecifiedComponents</partinfo><br />
|-<br />
! 5<sup>vec</sup><br />
| pGA15<br />
| [http://partsregistry.org/Part:BBa_I739205 BBa_I739205]<br />
| GENEART cloning vector, KmR<br><br />
| <partinfo>BBa_I739205 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Cell Strains'''<br />
|-<br />
|-<br />
!width="111"| 1<sup>str</sup><br />
|width="285"| Top10<br />
| [http://partsregistry.org/Part:BBa_V1009 BBa_V1009]<br />
|width="250"| chemically competent E.coli<br>from Invitrogen<br />
|width="157"| <partinfo>BBa_V1009 SpecifiedComponents</partinfo><br />
|-<br />
! 2<sup>str</sup><br />
| JM101<br />
| [http://partsregistry.org/Part:BBa_I739301 BBa_I739301]<br />
| original blue/white cloning strain<br />
| <partinfo>BBa_I739301 SpecifiedComponents</partinfo><br />
|}<br />
<br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-24T18:59:08Z<p>Kdikaiou: Final (?) changes</p>
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__NOTOC__<br />
<br />
= Introduction =<br />
<br />
On this page, you can find an analysis of the function of our system, its biological design, and a list of the parts that make up the system. Under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered.<br />
<br />
EducatETH <i>E.coli</i> is a system which can distinguish between [http://openwetware.org/wiki/ATc anhydrotetracycline (aTc)] and [http://openwetware.org/wiki/IPTG Isopropyl-beta-D-thiogalactopyranoside (IPTG)] based on a previous learning phase conducted with the same chemicals and the help of [http://partsregistry.org/Acyl-HSLs AHL]. It is composed of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (LacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists an extended version of the original toggle switch found in [1]. That is, a multi-inducible toggle switch. The main difference is reflected in the use of double promoters, so that the toggle switch only changes its state when both, one of the two chemicals (aTc/IPTG), and AHL are present. As AHL is only present during the learning phase, the toggle maintains its state during testing/recognition, and thus can “memorize”. AHL can therefore be seen as a training- or learning substance. In the reporting subsystem, four reporters ([http://partsregistry.org/Featured_Parts:Fluorescent_proteins fluorescent proteins]) allow supervision of (1.) the chemical the system was trained with and (2.) if the system recognizes the chemical it is being exposed to in the recognition phase as one it has been previously trained with or not.<br />
<br />
== The Complete System ==<br />
<br />
<p>[[Image:Biol_system_stand20.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] The biological design of educatETH <i>E.coli</i> is presented in Fig. 1 and below, we clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modeled? Then visit the [[ETHZ/Model| System Modeling]]!)</p><br />
<br />
==== Constitutive Subsystem ====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). For now, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and LacI parts behave similarly: more specifically, the TetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus p22cII is produced). Correspondingly, cI is produced when IPTG is present.</p><br />
<br />
==== Learning Subsystem ====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has in turn been induced by aTc). The LuxR-AHL complex induces cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). In analogy to the upper part, the LuxR-AHL complex induces production of p22cII and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI in the system, which in turn was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
==== Reporting Subsystem ====<br />
<br />
<p>There are four reporters in the system. CFP (more precisely: enhanced CFP, that is ECFP) and YFP (more precisely: enhanced YFP, that is EYFP) are active during the learning phase of the system and show which chemical the system is exposed to during learning, whereas all four reporters (the latter and GFP and RFP) are active during the recognition phase and show if the system is exposed to the same chemical as in learning or not. <br />
More specifically, the YFP production is regulated with help of two operator sites controlled by cI and aTc (TetR inhibitor). cI inhibits the YFP production and aTc induces it. Therefore, YFP is synthesized when the system is exposed to only aTc and cI is not produced within the system (i.e. the system has not been previously exposed to IPTG). The production of the other fluorescent proteins is regulated in a similar manner. Overall, the production of the fluorescent proteins is regulated as follows:<br />
*YFP gets produced when the system is exposed to only aTc and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*CFP gets produced when the system is exposed to only IPTG and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).<br />
*GFP gets produced when the system is exposed to only IPTG and no cI is produced (i.e. the system has ''not'' been previously exposed to IPTG).<br />
*RFP gets produced when the system is exposed to only aTc and no p22cII is produced (i.e. the system has ''not'' been previously exposed to aTc).</p><br />
<br />
This behaviour is visualized in Fig. 1.<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== System Phases ==<br />
<br />
<p>The system operation is divided into three main phases: a learning phase, a memory phase and a recognition phase. During the learning phase, the system is first exposed to one of the two chemicals it is designed to detect (aTc or IPTG). During the memory phase, the specific chemical (aTc or IPTG) is removed and AHL is added to activate the systems internal toggle switch. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was detected) or CFP (if IPTG was detected). During the recognition phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. Lets compare the systems toggle switch state with the effect of the newly introduced chemical: the system shows a different response if it has previously been exposed to this certain chemical and reports with the same XFP as in the learning phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc). The following table represents all possible paths that may be taken by the system during all phases of operation according to external stimuli: </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
!width="44" style="background:#446084; color:white"| aTc<br />
!width="44" style="background:#446084; color:white"| IPTG<br />
!width="44" style="background:#446084; color:white"| AHL<br />
!width="44" style="background:#446084; color:white"| p22cII<br />
!width="44" style="background:#446084; color:white"| cI<br />
! style="background:#446084; color:white"| Reporting <br />
|- <br />
|colspan="7" style="background:#96c9cf;" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| non<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP (fading)<br>finally no color<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP (fading)<br>finally no color<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
== System Parts ==<br />
<br />
<p>educatETH <i>E.coli</i> was implemented with 11 parts designed by the ETH Zurich team. [hhttps://2007.igem.org/wiki/index.php?title=ETHZ/Biology&action=submit The list of all the parts used] is available. Because the part information is retrieved from the Registry, the page needs some time to load. <br>(Are you interested in this information because you want to implement educatETH <i>E.coli</i> in your lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!)</p><br />
<br />
== References ==<br />
<p><br />
[http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html &#91;1&#93; Gardner TS, Cantor CR and Collins JJ] <i>"Construction of a genetic toggle switch in Escherichia coli"</i>, Nature 403:339–342, 2000<br /><br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:red;>Katerina's note: Put Sylke's new scheme on this page. Note: the scheme is ready, it just needs to be uploaded.</span><br />
<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-24T18:44:00Z<p>Kdikaiou: Corrected "exposed to p22..."</p>
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__NOTOC__<br />
<br />
= Introduction =<br />
<br />
On this page, you can find an analysis of the function of our system, its biological design, and a list of the parts that make up the system. Under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered.<br />
<br />
EducatETH <i>E.coli</i> is a system which can distinguish between [http://openwetware.org/wiki/ATc anhydrotetracycline (aTc)] and [http://openwetware.org/wiki/IPTG Isopropyl-beta-D-thiogalactopyranoside (IPTG)] based on a previous learning phase conducted with the same chemicals and the help of [http://partsregistry.org/Acyl-HSLs AHL]. It is composed of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (LacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists an extended version of the original toggle switch found in [1]. That is, a multi-inducible toggle switch. The main difference is reflected in the use of double promoters, so that the toggle switch only changes its state when both, one of the two chemicals (aTc/IPTG), and AHL are present. As AHL is only present during the learning phase, the toggle maintains its state during testing/recognition, and thus can “memorize”. AHL can therefore be seen as a training- or learning substance. In the reporting subsystem, four reporters ([http://partsregistry.org/Featured_Parts:Fluorescent_proteins fluorescent proteins]) allow supervision of (1.) the chemical the system was trained with and (2.) if the system recognizes the chemical it is being exposed to in the recognition phase as one it has been previously trained with or not.<br />
<br />
== The Complete System ==<br />
<br />
<p>[[Image:Biol_system_stand20.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] The biological design of educatETH <i>E.coli</i> is presented in Fig. 1 and below, we clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modeled? Then visit the [[ETHZ/Model| System Modeling]]!)</p><br />
<br />
==== Constitutive Subsystem ====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). For now, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and LacI parts behave similarly: more specifically, the TetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus p22cII is produced). Correspondingly, cI is produced when IPTG is present.</p><br />
<br />
==== Learning Subsystem ====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has in turn been induced by aTc). The LuxR-AHL complex induces cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). In analogy to the upper part, the LuxR-AHL complex induces production of p22cII and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI in the system, which in turn was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
==== Reporting Subsystem ====<br />
<br />
<p>There are four reporters in the system. CFP (more precisely: enhanced CFP, that is ECFP) and YFP (more precisely: enhanced YFP, that is EYFP) are active during the learning phase of the system and show which chemical the system is exposed to during learning, whereas all four reporters (the latter and GFP and RFP) are active during the recognition phase and show if the system is exposed to the same chemical as in learning or not. <br />
More specifically, the YFP production is regulated with help of two operator sites controlled by cI and aTc (TetR inhibitor). cI inhibits YFP production and aTc induces it. Therefore, YFP is synthesized when the system is exposed to only aTc and cI is not produced within the system. The production of the other fluorescent proteins is regulated in a similar manner:<br />
<br> CFP gets produced when the system is exposed to only IPTG and no p22cII is produced<br><br />
GFP gets produced when the system is exposed to only IPTG and no cI is produced<br><br />
RFP gets produced when the system is exposed to only aTc and no p22cII is produced</p><br><br />
<br />
== System Phases ==<br />
<br />
<p>The system operation is divided into [https://2007.igem.org/Image:ETHzFlowdiagram2.png three main phases]: a learning phase, a memory phase and a recognition phase. During the learning phase, the system is first exposed to one of the two chemicals it is designed to detect (aTc or IPTG). During the memory phase, the specific chemical (aTc or IPTG) is removed and AHL is added to activate the systems internal toggle switch. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was detected) or CFP (if IPTG was detected). During the recognition phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. Lets compare the systems toggle switch state with the effect of the newly introduced chemical: the system shows a different response if it has previously been exposed to this certain chemical and reports with the same XFP as in the learning phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc). The following table represents all possible paths that may be taken by the system during all phases of operation according to external stimuli: </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
!width="44" style="background:#446084; color:white"| aTc<br />
!width="44" style="background:#446084; color:white"| IPTG<br />
!width="44" style="background:#446084; color:white"| AHL<br />
!width="44" style="background:#446084; color:white"| p22cII<br />
!width="44" style="background:#446084; color:white"| cI<br />
! style="background:#446084; color:white"| Reporting <br />
|- <br />
|colspan="7" style="background:#96c9cf;" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| non<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP (fading)<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP (fading)<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
== System Parts ==<br />
<br />
<p>educatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids. A detailed list of the used parts and some background information can be found [[ETHZ/Biology/parts |'''here''']] (due to many pictures, it takes some time to load).</p><br />
<br />
== References ==<br />
<p><br />
[http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html &#91;1&#93; Gardner TS, Cantor CR and Collins JJ] <i>"Construction of a genetic toggle switch in Escherichia coli"</i>, Nature 403:339–342, 2000<br /><br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:blue;>Update and correct parts in parts list. Write better in a table</span><br />
<li> <span style=color:blue;>Update and correct full system scheme</span><br />
<li> <span style=color:blue;>What is the proof of concept mentioned? Is explained in the parts section!</span><br />
<li> <span style=color:blue;>Are you sure about the reporters? This is not what I understood. I though YFP and CFP were also during testing. Corrected!</span><br />
<li> <span style=color:blue;>I don't think it is wise to make p22cII in P22 cII, since we have in the first way all over the place. Is changed back!</span><br />
<li> <span style=color:red;>Try to improve table with system phases. It doesn't look so nice... </span><br />
<li> <span style=color:red;>In the memorizing phase, is there color or not? - Stefan: There are colors (due to the fact that there is still aTc or IPTG in the cell even after removing it), however they will disappear within 2-3 hours.</span><br />
<li> <span style=color:red;>We need to put the complete spaghetti system as well.</span><br />
<li> <span style=color:blue;>Check my terminology (operator sites etc). Checked!<br />
<li> <span style=color:blue;>Put Stefan's updated part on epigenetics. Is in the application section!<br />
<li> Fill in table completely, make it more reading-friendly<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/SimulationETHZ/Simulation2007-10-24T18:40:35Z<p>Kdikaiou: Corrected text in sensitivity analysis</p>
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__NOTOC__<br />
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=Introduction=<br />
<br />
As has been presented in the [[ETHZ/Model | Modeling]] page, we have created a model of our system that can be described as a finite state machine. In order to examine the behavior of our system thoroughly, we have to simulate it through all the difference phases that can be reached. We carry out our simulations based on the protocol presented in Fig. 1.<br />
<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== Simulation of Test Cases ==<br />
To test all the possible conditions and transitions in our system, we run timing simulations for all 4 different branches in the flow diagram of our protocol, and we plot the concentrations of the inducers, memory proteins and reporter proteins. During each learning, memorizing and recognizing phase, we wait for the steady state of the system to be reached. The three phases of the system are thus separated sufficiently in time. In all the cases, we follow the following procedure:<br />
* During the first 1000 minutes of the simulation, we do not add any inducers to the system, and as a result, we check for the baseline production of proteins.<br />
* Between 1000 and 2000 minutes, we add the inducer that should be learned, and we let the system reach steady state.<br />
* After 2000 minutes, we add the inducer AHL for memory formation, and run the simulation for an additional 1000 minutes, to reach again steady state.<br />
* Between 3000 and 4000 minutes, we test the final behavior of the system, and check if it reacts in the desired way, by showing the appropriate color.<br />
<br />
The parameters of the system are crucial if one wants to have accurate and realistic simulations. We present the parameters used to simulate our system, in the section [[ETHZ/Parameters | Parameters]].<br />
<br />
<p>[[Image:ETHZTest1_Stand24.10.png|left|thumb|<b>Fig. 2</b>: Test case 1 - Learn and recognize IPTG. The simulated behavior of the system, when IPTG is presented both during the learning phase, and the recognition phase. In both the learning and recognition phase, the system reports by producing CFP, which matches the desired behavior. |420px]]<br />
[[Image:ETHZTest2_Stand24.10.png|thumb|<b>Fig. 3</b>: Test case 2 - Learn IPTG and get tested for aTc. The simulated behavior of the system, when IPTG is presented during the learning phase, and aTc is presented during the recognition phase. The system reports by producing RFP during the recognition phase, which matches the desired behavior. |420px]]<br />
<br />
[[Image:ETHZTest4_Stand24.10.png|left|thumb|<b>Fig. 4</b>: Test case 3 - Learn aTc and get tested for IPTG. The simulated behavior of the system, when aTc is presented during the learning phase, and IPTG is presented during the recognition phase. The system reports by producing GFP during the recognition phase, which matches the desired behavior. |420px]]</p><br />
<br />
[[Image:ETHZTest3_Stand24.10.png|right|thumb|<b>Fig. 5</b>: Test case 4 - Learn and recognize aTc. The simulated behavior of the system, when aTc is presented both during the learning phase and the recognition phase. In both the learning and recognition phase the system reports by producing YFP, which matches the desired behavior. |420px]]<br />
<br />
==Sensitivity Analysis==<br />
<br />
Before performing simulations we did an intensive literature review to find the parameters of our system. Due to the uncertainty in parameter retrieval, we would like our system to have reduced sensitivity to the parameters. That way, we can have a guaranteed stable performance, even under non-simulated conditions.<br />
<br />
[[Image:ETHZSensitivity.png|right|thumb|<b>Fig. 6</b>: Sensitivity analysis. We plot the sensitivity for each parameter and each test case that corresponds to the different path ways in Fig. 1. |500px]]<br />
<br />
We define sensitivity as the change of the production of the desired florescence protein - which corresponds to the output of our system - depending on the change of the parameters. As shown in Fig. 1 the desired florescence protein output depends on the inducers that we present to our system during learning and recognition phase. Overall we have 4 different combinations leading to the 4 different test cases shown in Fig. 1. E.g. when IPTG is presented to the system both during the learning and recognition phase we expect the production of CFPs. That is why we define the sensitivity for test case 1 as<br />
<br />
[[Image:ETHZSensititivityequation.png|center|300px]]<br />
<br />
<!---<math>Sensitivity = \left | \frac{[ CFP^{105%}_{recognition} ] }{\left [ CFP^{105%}_{baseline}\right ] }- \frac{\left [ CFP^{95%}_{recognition} \right ] }{\left [ CFP^{95%}_{baseline} \right ] }\right |</math>---><br />
<br />
where <br />
* [CFP<sub>recognition</sub><sup>105%</sup>] describes the concentration of CFP during the recognition phase when the parameter that we test for is increased by 5%, <br />
* [CFP<sub>baseline</sub><sup>105%</sup>] describes the concentration of CFP without any inducers given to the system when the parameter that we test for is increased by 5%,<br />
* [CFP<sub>recognition</sub><sup>95%</sup>] describes the concentration of CFP during the recognition phase when the parameter that we test for is decreased by 5%, and <br />
* [CFP<sub>baseline</sub><sup>95%</sup>] describes the concentration of CFP without any inducers given to the system when the parameter that we test for is decreased by 5%.<br />
<br />
All concentrations are gained from timing simulations as shown in Fig. 2 when the concentrations reached steady state. E.g. [CFP<sub>recognition</sub>] corresponds to the value of CFP after 4000 minutes while [CFP<sub>baseline</sub>] is equal to the value for CFP after 1000 minutes.<br />
<br />
We define sensitivity by using the ratio of [CFP<sub>recognition</sub>] and [CFP<sub>baseline</sub>] to see by which factor the CFP concentration is increased during the recognition phase. The higher this value is the easier it will be possible to determine the correct readout.<br />
<br />
We subtract the two ratios of [CFP<sub>recognition</sub>]/[CFP<sub>baseline</sub>] for increasing and decreasing the parameter under test by 5% to quantify how much is the influence of the parameter on the output. If the output ratio does not change the parameter has no influence and the sensitivity is equal to zero.<br />
<br />
We quantified the sensitivity for all parameters and all 4 test cases. Therefor we look at the concentration of <br />
* CFP in test case 1. A timing simulation for this case is shown in Fig. 2.<br />
* RFP in test case 2. A timing simulation for this case is shown in Fig. 3.<br />
* GFP in test case 3. A timing simulation for this case is shown in Fig. 4.<br />
* YFP in test case 4. A timing simulation for this case is shown in Fig. 5.<br />
The results are plotted in Fig. 6. Furthermore we make sure that no undesired florescence protein is produced during the recognition phase.<br />
<br />
The conclusions that can be derived from our sensitivity analysis are the following:<br />
* Most of the parameters are not sensitive which corresponds to the desired behavior since the system should ideally give a constant output for the same input sequence.<br />
* Our system is most sensitive to the parameter "a", which describes the base production. This is not a surprising result, since we depend on having a good switch. However, we cannot reliably switch off the production of the memory proteins cI and p22cII. This leads to a small signal to noise ration and in the worst case the memory does not work at all. For a more detailed analysis of the switching behavior please see our [[ETHZ/Biology | biological implementation]] page.<br />
* Other sensitive parameters are those that are directly related to the production and decay of the memory proteins cI and p22cII. This can be explained by the fact that our system prefers symmetry between the parameter sets of cI and p22cII, in order to support good switching behavior where the same concentration of p22cII leads to the same repression of cI and vice versa. We can especially see that those parameters seem to be more sensitive for the test cases 1 and 2, where cI has to be stored inside the memory. The reason for this behavior is due to the missing symmetry between the parameters for cI and p22cII, we currently have a bias inside the system towards the production of p22cII.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/SimulationETHZ/Simulation2007-10-24T18:38:18Z<p>Kdikaiou: </p>
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__NOTOC__<br />
<br />
=Introduction=<br />
<br />
As has been presented in the [[ETHZ/Model | Modeling]] page, we have created a model of our system that can be described as a finite state machine. In order to examine the behavior of our system thoroughly, we have to simulate it through all the difference phases that can be reached. We carry out our simulations based on the protocol presented in Fig. 1.<br />
<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== Simulation of Test Cases ==<br />
To test all the possible conditions and transitions in our system, we run timing simulations for all 4 different branches in the flow diagram of our protocol, and we plot the concentrations of the inducers, memory proteins and reporter proteins. During each learning, memorizing and recognizing phase, we wait for the steady state of the system to be reached. The three phases of the system are thus separated sufficiently in time. In all the cases, we follow the following procedure:<br />
* During the first 1000 minutes of the simulation, we do not add any inducers to the system, and as a result, we check for the baseline production of proteins.<br />
* Between 1000 and 2000 minutes, we add the inducer that should be learned, and we let the system reach steady state.<br />
* After 2000 minutes, we add the inducer AHL for memory formation, and run the simulation for an additional 1000 minutes, to reach again steady state.<br />
* Between 3000 and 4000 minutes, we test the final behavior of the system, and check if it reacts in the desired way, by showing the appropriate color.<br />
<br />
The parameters of the system are crucial if one wants to have accurate and realistic simulations. We present the parameters used to simulate our system, in the section [[ETHZ/Parameters | Parameters]].<br />
<br />
<p>[[Image:ETHZTest1_Stand24.10.png|left|thumb|<b>Fig. 2</b>: Test case 1 - Learn and recognize IPTG. The simulated behavior of the system, when IPTG is presented both during the learning phase, and the recognition phase. In both the learning and recognition phase, the system reports by producing CFP, which matches the desired behavior. |420px]]<br />
[[Image:ETHZTest2_Stand24.10.png|thumb|<b>Fig. 3</b>: Test case 2 - Learn IPTG and get tested for aTc. The simulated behavior of the system, when IPTG is presented during the learning phase, and aTc is presented during the recognition phase. The system reports by producing RFP during the recognition phase, which matches the desired behavior. |420px]]<br />
<br />
[[Image:ETHZTest4_Stand24.10.png|left|thumb|<b>Fig. 4</b>: Test case 3 - Learn aTc and get tested for IPTG. The simulated behavior of the system, when aTc is presented during the learning phase, and IPTG is presented during the recognition phase. The system reports by producing GFP during the recognition phase, which matches the desired behavior. |420px]]</p><br />
<br />
[[Image:ETHZTest3_Stand24.10.png|right|thumb|<b>Fig. 5</b>: Test case 4 - Learn and recognize aTc. The simulated behavior of the system, when aTc is presented both during the learning phase and the recognition phase. In both the learning and recognition phase the system reports by producing YFP, which matches the desired behavior. |420px]]<br />
<br />
==Sensitivity Analysis==<br />
<br />
Before performing simulations we did an intensive literature review to find the parameters of our system. Due to the uncertainty in parameter retrieval, we would like our system to have reduced sensitivity to the parameters. That way, we can have a guaranteed stable performance, even under non-simulated conditions.<br />
<br />
[[Image:ETHZSensitivity.png|right|thumb|<b>Fig. 6</b>: Sensitivity analysis. We plot the sensitivity for each parameter and each test case that corresponds to the different path ways in Fig. 1. |500px]]<br />
<br />
We define sensitivity as the change of the production of the desired florescence protein - which corresponds to the output of our system - depending on the change of the parameters. As shown in Fig. 1 the desired florescence protein output depends on the inducers that we present to our system during learning and recognition phase. Overall we have 4 different combinations leading to the 4 different test cases shown in Fig. 1. E.g. when IPTG is presented to the system both during the learning and recognition phase we expect the production of CFPs. That is why we define the sensitivity for test case 1 as<br />
<br />
[[Image:ETHZSensititivityequation.png|center|300px]]<br />
<br />
<!---<math>Sensitivity = \left | \frac{[ CFP^{105%}_{recognition} ] }{\left [ CFP^{105%}_{baseline}\right ] }- \frac{\left [ CFP^{95%}_{recognition} \right ] }{\left [ CFP^{95%}_{baseline} \right ] }\right |</math>---><br />
<br />
where <br />
* [CFP<sub>recognition</sub><sup>105%</sup>] describes the concentration of CFP during the recognition phase when the parameter that we test for is increased by 5%, <br />
* [CFP<sub>baseline</sub><sup>105%</sup>] describes the concentration of CFP without any inducers given to the system when the parameter that we test for is increased by 5%,<br />
* [CFP<sub>recognition</sub><sup>95%</sup>] describes the concentration of CFP during the recognition phase when the parameter that we test for is decreased by 5%, and <br />
* [CFP<sub>baseline</sub><sup>95%</sup>] describes the concentration of CFP without any inducers given to the system when the parameter that we test for is decreased by 5%.<br />
<br />
All concentrations are gained from timing simulations as shown in Fig. 2 when the concentrations reached steady state. E.g. [CFP<sub>recognition</sub>] corresponds to the value of CFP after 4000 minutes while [CFP<sub>baseline</sub>] is equal to the value for CFP after 1000 minutes.<br />
<br />
We define sensitivity by using the ratio of [CFP<sub>recognition</sub>] and [CFP<sub>baseline</sub>] to see by which factor the CFP concentration is increased during the recognition phase. The higher this value is the easier it will be possible to determine the correct readout.<br />
<br />
We subtract the two ratios of [CFP<sub>recognition</sub>]/[CFP<sub>baseline</sub>] for increasing and decreasing the parameter under test by 5% to quantify how much is the influence of the parameter on the output. If the output ratio does not change the parameter has no influence and the sensitivity is equal to zero.<br />
<br />
We quantified the sensitivity for all parameters and all 4 test cases. Therefor we look at the concentration of <br />
* CFP in test case 1. A timing simulation for this case is shown in Fig. 2.<br />
* RFP in test case 2. A timing simulation for this case is shown in Fig. 3.<br />
* YFP in test case 3. A timing simulation for this case is shown in Fig. 4.<br />
* GFP in test case 4. A timing simulation for this case is shown in Fig. 5.<br />
The results are plotted in Fig. 6. Furthermore we make sure that no undesired florescence protein is produced during the recognition phase.<br />
<br />
The conclusions that can be derived from our sensitivity analysis are the following:<br />
* Most of the parameters are not sensitive which corresponds to the desired behavior since the system should ideally give a constant output for the same input sequence.<br />
* Our system is most sensitive to the parameter "a", which describes the base production. This is not a surprising result, since we depend on having a good switch. However, we cannot reliably switch off the production of the memory proteins cI and p22cII. This leads to a small signal to noise ration and in the worst case the memory does not work at all. For a more detailed analysis of the switching behavior please see our [[ETHZ/Biology | biological implementation]] page.<br />
* Other sensitive parameters are those that are directly related to the production and decay of the memory proteins cI and p22cII. This can be explained by the fact that our system prefers symmetry between the parameter sets of cI and p22cII, in order to support good switching behavior where the same concentration of p22cII leads to the same repression of cI and vice versa. We can especially see that those parameters seem to be more sensitive for the test cases 1 and 2, where cI has to be stored inside the memory. The reason for this behavior is due to the missing symmetry between the parameters for cI and p22cII, we currently have a bias inside the system towards the production of p22cII.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest4_Stand24.10.pngFile:ETHZTest4 Stand24.10.png2007-10-24T18:37:17Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest3_Stand24.10.pngFile:ETHZTest3 Stand24.10.png2007-10-24T18:36:59Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest2_Stand24.10.pngFile:ETHZTest2 Stand24.10.png2007-10-24T18:36:43Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest1_Stand24.10.pngFile:ETHZTest1 Stand24.10.png2007-10-24T18:36:22Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/SimulationETHZ/Simulation2007-10-24T18:36:02Z<p>Kdikaiou: Changed figures to correct ones</p>
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<br />
=Introduction=<br />
<br />
As has been presented in the [[ETHZ/Model | Modeling]] page, we have created a model of our system that can be described as a finite state machine. In order to examine the behavior of our system thoroughly, we have to simulate it through all the difference phases that can be reached. We carry out our simulations based on the protocol presented in Fig. 1.<br />
<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |500px]]<br />
<br />
== Simulation of Test Cases ==<br />
To test all the possible conditions and transitions in our system, we run timing simulations for all 4 different branches in the flow diagram of our protocol, and we plot the concentrations of the inducers, memory proteins and reporter proteins. During each learning, memorizing and recognizing phase, we wait for the steady state of the system to be reached. The three phases of the system are thus separated sufficiently in time. In all the cases, we follow the following procedure:<br />
* During the first 1000 minutes of the simulation, we do not add any inducers to the system, and as a result, we check for the baseline production of proteins.<br />
* Between 1000 and 2000 minutes, we add the inducer that should be learned, and we let the system reach steady state.<br />
* After 2000 minutes, we add the inducer AHL for memory formation, and run the simulation for an additional 1000 minutes, to reach again steady state.<br />
* Between 3000 and 4000 minutes, we test the final behavior of the system, and check if it reacts in the desired way, by showing the appropriate color.<br />
<br />
The parameters of the system are crucial if one wants to have accurate and realistic simulations. We present the parameters used to simulate our system, in the section [[ETHZ/Parameters | Parameters]].<br />
<br />
<p>[[Image:ETHZTest1_Stand24.10.png|left|thumb|<b>Fig. 2</b>: Test case 1 - Learn and recognize IPTG. The simulated behavior of the system, when IPTG is presented both during the learning phase, and the recognition phase. In both the learning and recognition phase, the system reports by producing CFP, which matches the desired behavior. |420px]]<br />
[[Image:ETHZTest2_Stand24.10.png|thumb|<b>Fig. 3</b>: Test case 2 - Learn IPTG and get tested for aTc. The simulated behavior of the system, when IPTG is presented during the learning phase, and aTc is presented during the recognition phase. The system reports by producing RFP during the recognition phase, which matches the desired behavior. |420px]]<br />
<br />
[[Image:ETHZTest3_Stand24.10.png|left|thumb|<b>Fig. 4</b>: Test case 3 - Learn aTc and get tested for IPTG. The simulated behavior of the system, when aTc is presented during the learning phase, and IPTG is presented during the recognition phase. The system reports by producing GFP during the recognition phase, which matches the desired behavior. |420px]]</p><br />
<br />
[[Image:ETHZTest4_Stand24.10.png|right|thumb|<b>Fig. 5</b>: Test case 4 - Learn and recognize aTc. The simulated behavior of the system, when aTc is presented both during the learning phase and the recognition phase. In both the learning and recognition phase the system reports by producing YFP, which matches the desired behavior. |420px]]<br />
<br />
==Sensitivity Analysis==<br />
<br />
Before performing simulations we did an intensive literature review to find the parameters of our system. Due to the uncertainty in parameter retrieval, we would like our system to have reduced sensitivity to the parameters. That way, we can have a guaranteed stable performance, even under non-simulated conditions.<br />
<br />
[[Image:ETHZSensitivity.png|right|thumb|<b>Fig. 6</b>: Sensitivity analysis. We plot the sensitivity for each parameter and each test case that corresponds to the different path ways in Fig. 1. |500px]]<br />
<br />
We define sensitivity as the change of the production of the desired florescence protein - which corresponds to the output of our system - depending on the change of the parameters. As shown in Fig. 1 the desired florescence protein output depends on the inducers that we present to our system during learning and recognition phase. Overall we have 4 different combinations leading to the 4 different test cases shown in Fig. 1. E.g. when IPTG is presented to the system both during the learning and recognition phase we expect the production of CFPs. That is why we define the sensitivity for test case 1 as<br />
<br />
[[Image:ETHZSensititivityequation.png|center|300px]]<br />
<br />
<!---<math>Sensitivity = \left | \frac{[ CFP^{105%}_{recognition} ] }{\left [ CFP^{105%}_{baseline}\right ] }- \frac{\left [ CFP^{95%}_{recognition} \right ] }{\left [ CFP^{95%}_{baseline} \right ] }\right |</math>---><br />
<br />
where <br />
* [CFP<sub>recognition</sub><sup>105%</sup>] describes the concentration of CFP during the recognition phase when the parameter that we test for is increased by 5%, <br />
* [CFP<sub>baseline</sub><sup>105%</sup>] describes the concentration of CFP without any inducers given to the system when the parameter that we test for is increased by 5%,<br />
* [CFP<sub>recognition</sub><sup>95%</sup>] describes the concentration of CFP during the recognition phase when the parameter that we test for is decreased by 5%, and <br />
* [CFP<sub>baseline</sub><sup>95%</sup>] describes the concentration of CFP without any inducers given to the system when the parameter that we test for is decreased by 5%.<br />
<br />
All concentrations are gained from timing simulations as shown in Fig. 2 when the concentrations reached steady state. E.g. [CFP<sub>recognition</sub>] corresponds to the value of CFP after 4000 minutes while [CFP<sub>baseline</sub>] is equal to the value for CFP after 1000 minutes.<br />
<br />
We define sensitivity by using the ratio of [CFP<sub>recognition</sub>] and [CFP<sub>baseline</sub>] to see by which factor the CFP concentration is increased during the recognition phase. The higher this value is the easier it will be possible to determine the correct readout.<br />
<br />
We subtract the two ratios of [CFP<sub>recognition</sub>]/[CFP<sub>baseline</sub>] for increasing and decreasing the parameter under test by 5% to quantify how much is the influence of the parameter on the output. If the output ratio does not change the parameter has no influence and the sensitivity is equal to zero.<br />
<br />
We quantified the sensitivity for all parameters and all 4 test cases. Therefor we look at the concentration of <br />
* CFP in test case 1. A timing simulation for this case is shown in Fig. 2.<br />
* RFP in test case 2. A timing simulation for this case is shown in Fig. 3.<br />
* YFP in test case 3. A timing simulation for this case is shown in Fig. 4.<br />
* GFP in test case 4. A timing simulation for this case is shown in Fig. 5.<br />
The results are plotted in Fig. 6. Furthermore we make sure that no undesired florescence protein is produced during the recognition phase.<br />
<br />
The conclusions that can be derived from our sensitivity analysis are the following:<br />
* Most of the parameters are not sensitive which corresponds to the desired behavior since the system should ideally give a constant output for the same input sequence.<br />
* Our system is most sensitive to the parameter "a", which describes the base production. This is not a surprising result, since we depend on having a good switch. However, we cannot reliably switch off the production of the memory proteins cI and p22cII. This leads to a small signal to noise ration and in the worst case the memory does not work at all. For a more detailed analysis of the switching behavior please see our [[ETHZ/Biology | biological implementation]] page.<br />
* Other sensitive parameters are those that are directly related to the production and decay of the memory proteins cI and p22cII. This can be explained by the fact that our system prefers symmetry between the parameter sets of cI and p22cII, in order to support good switching behavior where the same concentration of p22cII leads to the same repression of cI and vice versa. We can especially see that those parameters seem to be more sensitive for the test cases 1 and 2, where cI has to be stored inside the memory. The reason for this behavior is due to the missing symmetry between the parameters for cI and p22cII, we currently have a bias inside the system towards the production of p22cII.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest4correct.pngFile:ETHZTest4correct.png2007-10-22T15:53:55Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest3correct.pngFile:ETHZTest3correct.png2007-10-22T15:53:34Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest2correct.pngFile:ETHZTest2correct.png2007-10-22T15:53:14Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:ETHZTest1correct.pngFile:ETHZTest1correct.png2007-10-22T15:52:57Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/SimulationETHZ/Simulation2007-10-22T15:52:42Z<p>Kdikaiou: Changed figures to the correct ones.</p>
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<br />
=Introduction=<br />
<br />
As has been presented in the [[ETHZ/Model | Modeling]] page, we have created a model of our system that can be described as a finite state machine. In order to examine the behavior of our system thoroughly, we have to simulate it through all the difference phases that can be reached. We carry out our simulations based on the protocol presented in Fig. 1.<br />
<br />
[[Image:ETHzFlowdiagram2.png|center|thumb|<b>Fig. 1</b>: Flow diagram. This figure shows the protocol with which the final system should be tested, as well as the test results in the form of the reported colors. There are three phases the system has to go through: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and, (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input. |380px]]<br />
<br />
== Simulation of Test Cases ==<br />
To test all the possible conditions and transitions in our system, we run timing simulations for all 4 different branches in the flow diagram of our protocol, and we plot the concentrations of the inducers, memory proteins and reporter proteins. During each learning, memorizing and recognizing phase, we wait for the steady state of the system to be reached. The three phases of the system are thus separated sufficiently in time. In all the cases, we follow the following procedure:<br />
* During the first 1000 minutes of the simulation, we do not add any inducers to the system, and as a result, we check for the baseline production of proteins.<br />
* Between 1000 and 2000 minutes, we add the inducer that should be learned, and we let the system reach steady state.<br />
* After 2000 minutes, we add the inducer AHL for memory formation, and run the simulation for an additional 1000 minutes, to reach again steady state.<br />
* Between 3000 and 4000 minutes, we test the final behavior of the system, and check if it reacts in the desired way, by showing the appropriate color.<br />
<br />
The parameters of the system are crucial if one wants to have accurate and realistic simulations. We present the parameters used to simulate our system, in the section [[ETHZ/Parameters | Parameters]].<br />
<br />
<p>[[Image:ETHZTest1correct.png|left|thumb|<b>Fig. 2</b>: Test case 1 - Learn and recognize IPTG. The simulated behavior of the system, when IPTG is presented both during the learning phase, and the recognition phase. In both the learning and recognition phase, the system reports by producing cyan florescent proteins (CFP), which matches the desired behavior. |420px]]<br />
[[Image:ETHZTest2correct.png|thumb|<b>Fig. 3</b>: Test case 2 - Learn IPTG and get tested for aTc. The simulated behavior of the system, when IPTG is presented during the learning phase, and is presented aTc during the recognition phase. The system reports by producing red fluorescent proteins (RFP) during the recognition phase, which matches the desired behavior. |420px]]<br />
<br />
[[Image:ETHZTest3correct.png|left|thumb|<b>Fig. 4</b>: Test case 3 - Learn and recognize aTc. The simulated behavior of the system, when aTc is presented both during the learning phase and the recognition phase. In both the learning and recognition phase the system reports by producing yellow fluorescent proteins (YFP), which matches the desired behavior. |420px]]<br />
[[Image:ETHZTest4correct.png|right|thumb|<b>Fig. 5</b>: Test case 4 - Learn aTc and get tested for IPTG. The simulated behavior of the system, when aTc is presented during the learning phase, and IPTG is presented during the recognition phase. The system reports by producing green florescent proteins (GFP) during the recognition phase, which matches the desired behavior. |420px]]</p><br />
<br />
==Sensitivity Analysis==<br />
<br />
In order for one to ensure a biological system will operate as it was modeled, he/she should understand the sensitivity of the system's behavior to all involved parameters. Due to the uncertainty in parameter retrieval, we would like our system to have reduced sensitivity to the parameters that are affected mostly by noise. That way, we can have a guaranteed stable performance, even under non-simulated conditions.<br />
<br />
[[Image:ETHZSensitivity.png|right|thumb|<b>Fig. 5</b>: Sensitivity analysis |420px]]<br />
<br />
We simulated the behavior of our system throughout all its phases, while sweeping the parameters by 5%, one at a time. More specifically for the case presented in Fig. 1, we carried out the following procedure:<br />
For each parameter, we did the timing simulation as shown in Fig. 2. Once we increased the parameter by 5%, and once we decreased the parameter by 5%. Since in test case 1 (Fig. 2) we are interested in having a high concentration of CFP during the recognition phase with respect to the base line, we divide the steady state CFP concentration at 4000 minutes by the baseline concentration at 1000 minutes. Finally, as shown in the following equation, we define the sensitivity by subtracting the resulting factor that we get with an increased parameter, from the resulting factor that we get by a decreased parameter.<br />
<br />
[[Image:ETHZSensititivityequation.png|center|320px]]<br />
<br />
We can summarize that, if there is no difference in the concentration of the desired output during the recognition phase with respect to baseline when sweeping a parameter by +/-5%, the sensitivity of that parameter is equal to zero. Otherwise the sensitivity gives us a measure of how much the concentration changes.<br />
<br />
We quantified the sensitivity for all parameters and test cases. The results are plotted in Fig. 5.<br />
<br />
The conclusions that can be derived from our sensitivity analysis are the following:<br />
* Our system is most sensitive to the parameter "a", which describes the base production. This is not a surprising result, since we depend on having a good switch, and our signal-to-noise ratio, or output to baseline production level, depends highly on the fact that we can reliably switch off the production of the memory proteins cI and p22cII. For a more detailed analysis of the switching behavior please see our [[ETHZ/Biology | biological implementation]] page.<br />
* Other sensitive parameters are those that are directly related to the production and decay of the memory proteins cI and p22cII. This can be explained by the fact that our system prefers symmetry between the parameter sets of cI and p22cII, in order to support good switching behavior where the same concentration of p22cII leads to the same repression of cI and vice versa. We can especially see that those parameters seem to be more sensitive for the test cases 1 and 2, where cI has to be stored inside the memory. The reason for this behavior is due to the missing symmetry between the parameters for cI and p22cII, we currently have a bias inside the system towards the production of p22cII.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-20T16:10:00Z<p>Kdikaiou: Changed colours in Table with system phases</p>
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<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing educatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested in how educatETH ''E.coli'' was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
== Introduction ==<br />
<br />
<p> educatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
== The complete system ==<br />
<br />
<p>The biological design of educatETH <i>E.coli</i> is presented on [[Image:Biol_system_stand20.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=== Constitutive subsystem ===<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=== Learning subsystem ===<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=== Reporting subsystem ===<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
== System phases ==<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical and reports with the same XFP as in the training phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
!width="44" style="background:#446084; color:white"| aTc<br />
!width="44" style="background:#446084; color:white"| IPTG<br />
!width="44" style="background:#446084; color:white"| AHL<br />
!width="44" style="background:#446084; color:white"| p22cII<br />
!width="44" style="background:#446084; color:white"| cI<br />
! style="background:#446084; color:white"| Reporting <br />
|- <br />
|colspan="7" style="background:#96c9cf;" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" style="background:#96c9cf;" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
=== Further thoughts on the system phases ===<br />
<br />
TODO: put Stefan's new text here. Text should be small.<br />
<br />
== System parts ==<br />
<br />
educatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1<br />
|width="285"| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Composite System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1+2+3<br />
|width="285"| tetR + lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739013 SpecifiedComponents</partinfo><br />
|-<br />
! 4+5<br />
| P22 cII + EYFP production <br />
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo><br />
|-<br />
! 8+9<br />
| cI + ECFP production <br />
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo><br />
|-<br />
! (4+5)+(8+9)<br />
| (P22 cII + EYFP) + (cI + ECFP) production <br />
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo><br />
|-<br />
! 6+7<br />
| cI + P22 cII production <br />
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]<br />
| learning subsystem<br />
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo><br />
|-<br />
! 10+11<br />
| RFP + GFP production <br />
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo><br />
|-<br />
! (6+7)+(10+11)<br />
| (cI + P22 cII) + (RFP + GFP) production <br />
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]<br />
| learning/reporting subsystem<br />
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo><br />
|}<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
!width="111"| 1'<br />
|width="285"| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
|width="250"| reporting subsystem<br />
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2'<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3'<br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4'<br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5'<br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6'<br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
!width="111"| 1"<br />
|width="285"| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
|width="250"| proof of concept, no part of the system <br />
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2"<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|-<br />
! 3"<br />
| PoC composite<br />
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
== References ==<br />
<br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:blue;>Update and correct parts in parts list. Write better in a table</span><br />
<li> <span style=color:blue;>Update and correct full system scheme</span><br />
<li> <span style=color:red;>What is the proof of concept mentioned?</span><br />
<li> <span style=color:red;>Try to improve table with system phases. It doesn't look so nice...</span><br />
<li> <span style=color:red;>In the memorizing phase, is there color or not?</span><br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> Fill in table completely, make it more reading-friendly<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-20T09:55:02Z<p>Kdikaiou: Fixed table, todo's, text.</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing educatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested in how educatETH ''E.coli'' was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
== Introduction ==<br />
<br />
<p> educatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
== The complete system ==<br />
<br />
<p>The biological design of educatETH <i>E.coli</i> is presented on [[Image:Biol_system_stand20.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=== Constitutive subsystem ===<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=== Learning subsystem ===<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=== Reporting subsystem ===<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
== System phases ==<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical and reports with the same XFP as in the training phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''System phases''' <br />
! <br />
! aTc<br />
! IPTG<br />
! AHL<br />
! p22cII<br />
! cI<br />
! Reporting <br />
|- <br />
|colspan="7" align="center"|'''Start''' <br />
|- <br />
| no input<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| colspan="7" align="center"| '''Learning'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" align="center"| '''Memorizing'''<br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| colspan="7" align="center"| '''Recognition'''<br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| YFP<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| GFP<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| CFP<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| RFP<br />
|- <br />
<br />
|}<br />
<br />
=== Further thoughts on the system phases ===<br />
<br />
TODO: put Stefan's new text here. Text should be small.<br />
<br />
<br />
== System parts ==<br />
<br />
educatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1<br />
|width="285"| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
|width="250"| constitutive subsystem<br />
| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Composite System Parts'''<br />
|-<br />
|-<br />
! 1+2+3<br />
|width="285"| tetR + lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739013 SpecifiedComponents</partinfo><br />
|-<br />
! 4+5<br />
| P22 cII + EYFP production <br />
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo><br />
|-<br />
! 8+9<br />
| cI + ECFP production <br />
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo><br />
|-<br />
! (4+5)+(8+9)<br />
| (P22 cII + EYFP) + (cI + ECFP) production <br />
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo><br />
|-<br />
! 6+7<br />
| cI + P22 cII production <br />
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]<br />
| learning subsystem<br />
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo><br />
|-<br />
! 10+11<br />
| RFP + GFP production <br />
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo><br />
|-<br />
! (6+7)+(10+11)<br />
| (cI + P22 cII) + (RFP + GFP) production <br />
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]<br />
| learning/reporting subsystem<br />
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo><br />
|}<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
!width="111"| 1'<br />
| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
|width="250"| reporting subsystem<br />
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2'<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3'<br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4'<br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5'<br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6'<br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
!width="111"| 1"<br />
|width="285"| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
| proof of concept, no part of the system <br />
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2"<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|-<br />
! 3"<br />
| PoC composite<br />
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
== References ==<br />
<br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:blue;>Update and correct parts in parts list. Write better in a table</span><br />
<li> <span style=color:blue;>Update and correct full system scheme</span><br />
<li> <span style=color:red;>What is the proof of concept mentioned?</span><br />
<li> <span style=color:red;>Try to improve table with system phases. It doesn't look so nice...</span><br />
<li> <span style=color:red;>In the memorizing phase, is there color or not?</span><br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> Fill in table completely, make it more reading-friendly<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-20T08:52:10Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing educatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested in how educatETH ''E.coli'' was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
== Introduction ==<br />
<br />
<p> educatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
== The complete system ==<br />
<br />
<p>The biological design of educatETH <i>E.coli</i> is presented on [[Image:Biol_system_stand20.10.png|thumb|left|300px|'''Fig. 1:''' Gene interaction network of educatETH ''E.coli'' ]] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=== Constitutive subsystem ===<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=== Learning subsystem ===<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=== Reporting subsystem ===<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
== System phases ==<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical and reports with the same XFP as in the training phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! <br />
! aTc<br />
! IPTG<br />
! AHL<br />
! p22cII<br />
! cI<br />
! Reporting <br />
|- <br />
| '''Start'''<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| '''Learning'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| '''Seeing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Memorizing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Testing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| gfp<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| rfp<br />
|- <br />
<br />
|}<br />
<br />
=== Further thoughts on the system phases ===<br />
<br />
TODO: put Stefan's new text here. Text should be small.<br />
<br />
<br />
== System parts ==<br />
<br />
educatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1<br />
|width="285"| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
|width="250"| constitutive subsystem<br />
| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Composite System Parts'''<br />
|-<br />
|-<br />
! 1+2+3<br />
|width="285"| tetR + lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739013 SpecifiedComponents</partinfo><br />
|-<br />
! 4+5<br />
| P22 cII + EYFP production <br />
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo><br />
|-<br />
! 8+9<br />
| cI + ECFP production <br />
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo><br />
|-<br />
! (4+5)+(8+9)<br />
| (P22 cII + EYFP) + (cI + ECFP) production <br />
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo><br />
|-<br />
! 6+7<br />
| cI + P22 cII production <br />
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]<br />
| learning subsystem<br />
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo><br />
|-<br />
! 10+11<br />
| RFP + GFP production <br />
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo><br />
|-<br />
! (6+7)+(10+11)<br />
| (cI + P22 cII) + (RFP + GFP) production <br />
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]<br />
| learning/reporting subsystem<br />
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo><br />
|}<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
!width="111"| 1'<br />
| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
|width="250"| reporting subsystem<br />
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2'<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3'<br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4'<br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5'<br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6'<br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
!width="111"| 1"<br />
|width="285"| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
| proof of concept, no part of the system <br />
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2"<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|-<br />
! 3"<br />
| PoC composite<br />
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
== References ==<br />
<br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> <span style=color:blue;>Update and correct parts in parts list. Write better in a table</span><br />
<li> <span style=color:blue;>Update and correct full system scheme</span><br />
<li> <span style=color:red;>What are the “double promoters” mentioned?</span><br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> Fill in table completely, make it more reading-friendly<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:Biol_system_stand20.10.pngFile:Biol system stand20.10.png2007-10-20T08:45:52Z<p>Kdikaiou: Simplified biological design of EducatETH E.coli. Made by Sylke on 19.10.2007, uploaded by Katerina on 20.10.2007.
Note: the image depicts the correct (ordered to Geneart) version of the system, i.e. with the correct reporters.</p>
<hr />
<div>Simplified biological design of EducatETH E.coli. Made by Sylke on 19.10.2007, uploaded by Katerina on 20.10.2007.<br />
<br />
Note: the image depicts the correct (ordered to Geneart) version of the system, i.e. with the correct reporters.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-20T08:43:37Z<p>Kdikaiou: Image</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing educatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab Lab Notes], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested on how educatETH ''E.coli'' was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
== Introduction ==<br />
<br />
<p> educatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
== The complete system ==<br />
<br />
<p>The biological design of EducatETH <i>E.coli</i> is presented on [[Image:Biol_system_stand20.10.png|thumb|left|300px|'''Fig. 1: '''educatETH ''E.coli'' System]] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=== Constitutive subsystem ===<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=== Learning subsystem ===<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=== Reporting subsystem ===<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
== System phases ==<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical and reports with the same XFP as in the training phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! <br />
! aTc<br />
! IPTG<br />
! AHL<br />
! p22cII<br />
! cI<br />
! Reporting <br />
|- <br />
| '''Start'''<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| '''Learning'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| '''Seeing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Memorizing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Testing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| gfp<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| rfp<br />
|- <br />
<br />
|}<br />
<br />
=== Further thoughts on the system phases ===<br />
<br />
TODO: put Stefan's new text here. Text should be small.<br />
<br />
<br />
== System parts ==<br />
<br />
EducatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1<br />
|width="285"| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
|width="250"| constitutive subsystem<br />
| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Composite System Parts'''<br />
|-<br />
|-<br />
! 1+2+3<br />
|width="285"| tetR + lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739013 SpecifiedComponents</partinfo><br />
|-<br />
! 4+5<br />
| P22 cII + EYFP production <br />
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo><br />
|-<br />
! 8+9<br />
| cI + ECFP production <br />
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo><br />
|-<br />
! (4+5)+(8+9)<br />
| (P22 cII + EYFP) + (cI + ECFP) production <br />
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo><br />
|-<br />
! 6+7<br />
| cI + P22 cII production <br />
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]<br />
| learning subsystem<br />
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo><br />
|-<br />
! 10+11<br />
| RFP + GFP production <br />
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo><br />
|-<br />
! (6+7)+(10+11)<br />
| (cI + P22 cII) + (RFP + GFP) production <br />
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]<br />
| learning/reporting subsystem<br />
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo><br />
|}<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
!width="111"| 1'<br />
| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
|width="250"| reporting subsystem<br />
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2'<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3'<br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4'<br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5'<br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6'<br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
!width="111"| 1"<br />
|width="285"| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
| proof of concept, no part of the system <br />
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2"<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|-<br />
! 3"<br />
| PoC composite<br />
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
== References ==<br />
<br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> Update and correct parts in parts list. Write better in a table<br />
<li> Update and correct full system scheme<br />
<li> Update graph scheme (made by Stefan) using aTc, IPTG instead of it1,2 and ia1,2<br />
<li> Which reporters are active when? I think CFP and YFP are not active only during training. Change text if needed.<br />
<li> Proposed terminology: seeing, memorizing<br />
<li> What are GFP, RFP controlled by? Is the full system scheme correct there?<br />
<li> What are the “double promoters” mentioned?<br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> How was Sven’s standard notation on how to write differently proteins, dna, rna?<br />
<li> Fill in table completely, make it more reading-friendly<br />
<li> Make "In the lab page", replace links.<br />
<li> Put image with 11 system parts (updated one, created by katerina)<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/LabETHZ/Biology/Lab2007-10-20T08:25:12Z<p>Kdikaiou: Changed "In the Lab" to "Lab Notes" to be consistent.</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|900px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - Lab Notes ::. </b></font></center><br><br />
<br />
In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section [https://2007.igem.org/ETHZ/Biology/Lab#.::_Problems_we_faced_::. Problems we faced] might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Biology | Biology Pespective]]. Finally, photos of our lab experience are accessible under [[ETHZ/Pictures | Pictures!]]<br />
<br />
Todo: decide what happens with lab book ([https://2007.igem.org/ETHZ/Lab_book here])<br />
<br />
==Cloning plan==<br />
<br />
===Parts assignment into plasmids===<br />
<br />
Three plasmids are used for the EducatETH <i>E.coli</i> system parts as follows:<br />
<br />
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|+'''Plasmids and contents'''<br />
|-<br />
! plasmid !! resistance !! copy type!! contents !! comments<br />
|-<br />
<br />
| [[ETHZ/pbr322| pbr322]] || ampicillin || high || 1,2,3 || constitutive subsystem<br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || chloramphenicol|| low || 4,5,8,9 || reporting subsystem<br />
|-<br />
<br />
| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| low || 6,7,10,11 || learning subsystem, reporting subsystem<br />
|-<br />
|}<br />
<br />
It is important to insert parts responsible for the production of fluorescent proteins in low copy plasmids, as they are potentially harmful for the cell. Unfortunately, working with low copy plasmids makes the procedure more demanding in the lab.<br />
<br />
===Linkers===<br />
<br />
Because the plasmids used were not standard plasmids found in the registry, but came from the lab where we work, linkers compatible with the standard BioBrick assembly have to be used in order to work with them. The list of all linkers is the following:<br />
<br />
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|+'''Linkers for plasmids'''<br />
|-<br />
! Linker!! Plasmid<br />
|-<br />
<br />
| [[ETHZ/pbr322-1| pbr322-1]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-2| pbr322-2]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-3| pbr322-3]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-4| pbr322-4]]|| pbr322 <br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || pck01 <br />
|-<br />
| [[ETHZ/pCK01-2| pck01-2]] || pck01 <br />
|-<br />
<br />
| [[ETHZ/pacyc177-1| pacyc177-1]] || pacyc177<br />
|-<br />
| [[ETHZ/pacyc177-2| pacyc177-2]]|| pacyc177<br />
|-<br />
|}<br />
<br />
Note that four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.<br />
<br />
=== Procedure ===<br />
<br />
The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. [[Image:Assembly _process.png|thumb|300px|DNA assembly process ([1]) '''(Fig. 4)''']] Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted ''before'' the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the ''donor'' and the plasmid accepting the new part will be referred to as the ''acceptor''. Composite pars made of parts '''a''' and '''b''' are denoted '''a.b'''.<br />
<br />
==== Plasmid 1 ''(pbr322ap)'' ====<br />
<br />
# Put parts 1,2,3 in pbr322ap plasmids. <br />
# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.<br />
# Merge plasmid containing part '''1''' ''(donor)'' with plasmid containing composite part '''2.3''' ''(acceptor)''. You should get a plasmid containing a '''1.2.3''' composite part.<br />
<br />
==== Plasmid 2 ''(pck01cm)''====<br />
<br />
# Put parts 4,5,8,9 in pck01cm plasmids. <br />
# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.<br />
# Merge plasmid containing part '''8''' ''(donor)'' with plasmid containing part '''9''' ''(acceptor)''. You should get a plasmid containing a '''8.9''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''4.5''' ''(donor)'' with plasmid containing composite part '''8.9''' ''(acceptor)''. You should get a plasmid containing a '''4.5.8.9''' composite part.<br />
<br />
====Plasmid 3 ''(pacyc177km)''====<br />
<br />
# Put parts 6,7,10,11 in pacyc177km plasmids. <br />
# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.<br />
# Merge plasmid containing part '''10''' ''(donor)'' with plasmid containing part '''11''' ''(acceptor)''. You should get a plasmid containing a '''10.11''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''6.7''' ''(donor)'' with plasmid containing composite part '''10.11''' ''(acceptor)''. You should get a plasmid containing a '''6.7.10.11''' composite part.<br />
<br />
== Problems we faced==<br />
<br />
== Lab book ==<br />
<br />
==== Week 1 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 06. Aug. 2007 <br />
|<br />
| <br />
* Preparing the Solutions <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Tue, 07. Aug. 2007 <br />
| <br />
|<br />
* Prepare competent cells for all parts<br />
* Transformation of all the parts <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Wed, 08. Aug. 2007 <br />
| <br />
| <br />
* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures) <br />
| Raphael<br>Stefan<br />
|-<br />
| Thu, 09. Aug. 2007 <br />
| <br />
| <br />
* MINIPREP of the grown (10) o/n cultures<br />
* Gelelectrophoresis of the grown cultures (step: 0.8% Agarose) <br />
| Raphael<br>Stefan<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Fri, 10. Aug. 2007 <br />
| <br />
| <br />
* 7 working parts/plasmids (step after "DIGESTS"): <br> (B0034, R0062, R0053, E0434, B0015, R0010, E0422)<br />
* 4 parts/plasmids minipreped: <br> (R0040, R0051, Q04121, C0053)<br />
|<br />
Christos <br> Markus <br> Stefan<br />
|-<br />
| Sat, 11. Aug. 2007 <br />
| <br />
| no labwork <br />
|<br />
|-<br />
| Sun, 12. Aug. 2007 <br />
|<br />
| labwork cancelled<br />
|<br />
|}<br />
<br />
==== Week 2 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 13. Aug. 2007 <br> start at 3 pm <br />
|<br />
* Prepare competent cells<br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053<br />
| <br />
* o/n culture (E.Coli Top10)<br />
* Control Restrictions (didn't work)<br />
| Martin<br> Markus <br> Christos <br> Tim <br><br />
|-<br />
| Tue, 14. Aug. 2007 <br />
| Morning Shift: <br><br />
* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br> <br />
Evening Shift: <br><br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
| Morning Shift: <br><br />
* Prepared competent cells (stored in the -80°C freezer in the basement) <br><br />
Evening Shift: <br><br />
* Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday) <br />
* Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4% <br />
|Morning Shift (9am-1pm?): <br> Markus <br> Tim <br> Evening Shift (5pm-...):<br> Martin <br> Christos<br />
|-<br />
| Wed, 15. Aug. 2007 <br />
| <br />
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434 <br />
|<br />
* Ligation didn't work due to bad quality of enzymes (probably) <br />
| From 12:<br> Martin<br>Markus<br><br><br />
|-<br />
| Thu, 16. Aug. 2007 <br />
| <br />
* Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)<br />
* Transformation of #13 and #14<br />
| <br />
* Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510) <br> One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)<br />
* Transformation of #13 (Q04121) and #14 (C0053) <br> Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)<br />
|<br />
Markus<br>Christos<br>(Martin)<br />
|-<br />
| Fri, 17. Aug. 2007 <br />
|<br />
* o/n of #13 and #14<br />
* Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful<br />
| <br />
* #13 and #14 didn't grow<br />
* # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates<br />
* New white pipette tips prepared (autoclave)<br />
* New bottles of Liquid LB and LB Agar prepared (autoclave)<br />
| Martin<br />
|-<br />
| Sat, 18. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 19. Aug. 2007 <br />
|<br />
|<br />
|<br />
|}<br />
<br />
==== Week 3 ====<br />
<br />
Little rearrangements of the parts. Planning of the sequences to order them.<br />
<br />
<br />
==== Week 4 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 27. Aug. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 28. Aug. 2007 <br />
| <br />
|<br />
| <br />
|-<br />
| Wed, 29. Aug. 2007 <br />
| <br />
| <br />
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|-<br />
| Thu, 30. Aug. 2007 <br />
| <br />
| <br />
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|-<br />
| Fri, 31. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 01. Sept. 2007 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 and plated them <br />
| Stefan<br />
|-<br />
| Sun, 02. Sept. 2007 <br />
|<br />
* Prepare o/n of pbr322, pcyc177, pck01<br />
| <br />
* o/n of pcyc177, pck01<br />
* the plates of pcyc177 and pck01 are in the fridge<br />
* transformed pbr322 because the culture didn't grow on the plate<br />
| Stefan<br />
|}<br />
<br />
==== Week 5 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 03. Sept. 2007 <br />
|<br />
* Prepare new competent cells<br />
* Miniprep pcyc177 and pcK01<br />
* prepare new o/n culture of pbr322<br />
* Run agarose gel of Minipreped plasmids<br />
| <br />
* New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)<br />
* Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)<br />
* pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday<br />
* new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids<br />
* 2 boxes of blue pipette tips are in the autoclave<br />
* Stefan ran the agarose gel (?) <br />
| Martin<br>Stefan<br />
|-<br />
| Tue, 04. Sept. 2007 <br />
|<br />
* Miniprep pcyc177 and pck01<br />
* cut the prepped plasmids to test if we've got the right ones<br />
* run agarose gel to test the cut and uncut ones <br />
* prepare new o/n of pbr322 (from Andy) <br />
|<br />
* Miniprep of pcyc177 and pck01 (but not yet tested)<br />
* Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep <br />
| Martin<br>Christian<br />
|-<br />
| Wed, 05. Sept. 2007 <br />
| <br />
* Miniprep of pbr322<br />
* Test-Digest of pcyc177 and pck01 and agarose gel...<br />
* Streak out all three plasmids on new plates, so we have them in reserve <br />
|<br />
* New Plate of pbr322.<br />
* Minipreps and Agarose Gels will be done tomorrow <br />
| Martin<br />
|-<br />
| Thu, 06. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01<br />
* Test with agarose gel <br />
| <br />
* Gel of the older plasmids -> plasmid present<br />
| Christian<br />
|-<br />
| Fri, 07. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01 <br />
| <br />
* Plasmids miniprepped<br />
| Martin<br />
|-<br />
| Sat, 08. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 09. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 6 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 10. Sept. 2007 <br />
| <br />
* Miniprep pBR322<br />
* annealing of different MCSs<br />
* Digest of pCK01 with BamHI+AseI<br />
* digest of pACYC177 with BamHI+PstI<br />
* digest of pBR322 with EcoRI+PstI <br />
all digests o/n<br />
| <br />
<br />
Christian<br />
| | <br />
|-<br />
| Tue, 11. Sept. 2007 <br />
|<br />
* Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work<br />
* 1x ligation of MCS inside backbones o/d, Trafo<br />
* 1x ligation of MCS inside backbones o/n<br />
| <br />
* plate all 3 plasmids for new minipreps<br />
|<br />
Christian<br />
|-<br />
| Wed, 12. Sept. 2007 <br />
|<br />
* Trafo of o/n ligations<br />
* o/n cultures of putative clones <br />
|<br />
|<br />
Christian<br />
|-<br />
| Thu, 13. Sept. 2007 <br />
| <br />
* Minipreps of putative clones pCK01-MCS and pBR322-MCS<br />
* control digests of putative clones<br />
* new o/n cultures of the putative clones of o/n ligations<br />
| <br />
| <br />
Christian<br />
|-<br />
| Fri, 14. Sept. 2007 <br />
| <br />
*separation of control digests of putative clones <br />
|<br />
'''*pBR322-MCS (Tet-selection) clone2 positive''' <br />
|<br />
Christian<br />
|-<br />
| Sat, 15. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 16. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 7 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 17. Sept. 2007 <br />
|<br />
* new digest of pACYC177 with BamHI+PstI o/n<br />
* digest of pACYC177, pBR322 AP<br />
* ligation of 177 and 322AP<br />
| <br />
* digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)<br />
* ligation of pBR322 o/n<br />
* 100 ml o/n culture to MAXIprep pacyc177<br />
* Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)<br />
| <br />
Christian <br> Martin, Raphael<br />
|-<br />
| Tue, 18. Sept. 2007 <br />
|<br />
* different control digests of pBR322-MCS (Tet) (see last week)<br />
* separation of pACYC177 digest<br />
* Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)<br />
* Transformation of the ligated pbr322 AP (MCS)<br />
* Prep pacyc177<br />
* Digest prepped pacyc177<br />
|<br />
> no DNA on pACYC177 digest-gel, only degradation smear<br> <br><br />
<br />
* Plates of pbr322 AP grew<br />
* No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)<br />
* Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )<br />
* Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.<br />
* Digest of pacyc177 with PstI o/n (pray that it will work!)<br />
* New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)<br />
* test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests<br />
| <br />
<br />
Christian <br><br />
Martin<br>Raphael<br />
|-<br />
| Wed, 19. Sept. 2007 <br />
| <br />
* o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated<br />
* Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)<br />
* check the digests of pacyc177 (pst) and pck01 (notI)<br />
* design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01 <br />
|<br />
|<br />
|-<br />
| Thu, 20. Sept. 2007 <br />
| <br />
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|-<br />
| Fri, 21. Sept. 2007 <br />
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| Sat, 22. Sept. 2007 <br />
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| Sun, 23. Sept. 2007 <br />
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|}<br />
<br />
==== Week 8 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 24. Sept. 2007 <br />
|<br />
| <br />
| <br />
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| Tue, 25. Sept. 2007 <br />
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| Wed, 26. Sept. 2007 <br />
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| Thu, 27. Sept. 2007 <br />
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| Fri, 28. Sept. 2007 <br />
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| Sat, 29. Sept. 2007 <br />
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| Sun, 30. Sept. 2007 <br />
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|}<br />
<br />
====Week 9 ====<br />
<br />
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! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 1. Oct. 2007 <br />
|<br />
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| Tue, 2. Oct. 2007 <br />
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| Wed, 3. Oct. 2007 <br />
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| Thu, 4. Oct. 2007 <br />
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| Fri, 5. Oct. 2007 <br />
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| Sat, 6. Oct. 2007 <br />
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|-<br />
| Sun, 7. Oct. 2007 <br />
|<br />
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|}<br />
<br />
==== Week 10 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 8. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 9. Oct. 2007 <br />
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|-<br />
| Wed, 10. Oct. 2007 <br />
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|<br />
|-<br />
| Thu, 11. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 12. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 13. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 14. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==References==<br />
<p>[1] [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly Process]</p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/InternalETHZ/Internal2007-10-20T08:24:11Z<p>Kdikaiou: Fixed headings, removed old task list (with dates etc)</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+2'><b> .:: ETH Zurich - educatETH ''E.coli'' ::. </b></font></center><br><br />
<br />
==Synthetic Biology Boot camp ==<br />
To make everybody familiar with synthetic biology concepts and to assist coming up with ideas for an interesting project, we will read and present publications on important topics. The list to be covered is the following: <br />
<br />
* Introduction to synthetic biology (Markus, Martin)<br />
* DNA de novo design (Rico, Raphael)<br />
* DNA circuits (Christian, Nan))<br />
* Hysteresis (Tim, Sylke)<br />
* Oscillators (Christos, Joe, Katerina and Markus, Martin)<br />
* Zinc fingers (Sylke)<br />
* Noise in single cell measurements<br />
* Distance communication (Rico, Raphael)<br />
* Parameter manipulations (Christos, Joe, Katerina)<br />
* Orthogonal systems (Christos, Joe, Katerina)<br />
* Minimal genomes (Markus, Martin)<br />
* RNA regulators (Rico, Raphael)<br />
<br />
All presentations have been uploaded in the known web space. You may also contact the people who did it if you need additional information.<br />
<br />
== Brainstorming ==<br />
<br />
=== Brainstorming sessions===<br />
During the first two weeks of July, the team has to come up with a project. The team is divided into three groups, which will brainstorm individually. Sven and Joerg are available on the 4th and 5th of July in case groups needs to consult them. The objective is that each group comes up with many fancy ideas. To facilitate this, keep in mind the following "brainstorming rules":<br />
<br />
# Defer judgment - the rules of nature don't apply<br />
# Encourage wild ideas<br />
# Build on the ideas of others<br />
# Be visual<br />
# Go for quantity<br />
# Stay focused on topic<br />
<br />
=== Preliminary ideas ===<br />
<br />
* '''PID Controller''': Design a PID controller out of biological elements. The P component can be a simple output to a regulatory protein, and the I component can be the overall protein production at a time period. What can the D component be?<br />
* '''Motion Detector''': Cells are grown on a petri dish. Below the dish, moving images are displayed. A 3-state automaton is proposed. Output A is created when light is present. Output B is created when light is absent. Moving patterns will cause some cells to create both outputs over time. This will result in some “inspector” cells producing output C, by collecting outputs A and B.<br />
* '''Analog-to-Digital Converter''': Compare the level of protein concentration with thresholds, and digitize the output.<br />
* '''Neural Network''': Create a sort of biological neural network with bacteria. We should address the issue of learning, and find a way to incorporate the feedback in the cell decision making process. Directed evolution can be a sort of feedback, but we want to avoid this. (This idea was the basis for the “learning project”)<br />
* '''Paramedic Cells''': Some cells are able to detect signals coming from other cells, and create food for them, or create proteins in order to save them and make them function better.<br />
* '''Cell Batteries''': Cells are able to create and store large quantities of ATP, during a “storing process”. Afterwards, they can detect a signal and give back all the energy they stored, in a short burst, like a capacitor. Other ideas are that the cells can “blow up” and emit large amounts of GFP, based on the ATP that they have accumulated.<br />
* '''Flashing Bacteria''': Cells are grown on a light pattern. The cells that are on the bright parts of the image are oscillating in phase, while the others are remaining dark. This results in the observation of a flashing pattern.<br />
* '''Biocam''': Visible to Fluorescent light converter.<br />
* '''BioCD''': “Print” cells on a film, then read them out and “reconstruct” the original data. Basically, it is an analog to digital converter, followed by a system that can interpret the digitized data. (This idea was the basis for the “Music of life project”, where cells would produce fluorescent proteins based on an analog input. Then, the amount and type of fluorescence would code some music).<br />
* '''Clock''': A follow-the-leader system. We have to groups of cells. The first group creates something that repels the second group. The second group creates a protein that attracts the first group. This way, they first group wants to “catch” the second group, whereas the second group wants to “avoid” the first group. This results in them moving around. We can say that the second group is the leader, and the first group exhibits a "follow-the-leader” behavior.<br />
* '''Sensors''': Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.<br />
<br />
=== Preferred projects===<br />
Three ideas of the above are chosen for further examination. The team will be again split up in three groups (different than before, to make sure that new ideas come up with mxing of people). Each group has to come up with an initial system, with remarks on its feasibility and coolness. Our results<br />
will be presented to all team members, so that potential projects may be limited down to two and subsequently to<br />
one. The preferred projects are: <br />
<br />
# '''Music of Life''': The basic idea is that instead of having an analog-to-digital converter with four outputs (three fluorescent proteins, and no output), we can have two switches. When switch A is on, RFP is produced. When switch B is on, GFP is produced. When both switches A and B are on, a yellowish output is observed. By recording these outputs, we can later create music, by assigning each fluorescent protein to a chord. For example, RFP would correspond to a G chord. The strength of the fluorescence can signify the strength of the chord. If the cells are placed on a spinning disk, we can have something like a vinyl player. A camera is observing the cells, and music is created on the fly. <br />
# '''Learning''': Based on the idea of the neural network, we want to create a biological system, where the cells can learn a specific behavior. In order to simplify the system, we decided that the cells can learn to recognize a specific type of other cells. We divide the process in a learning phase, and a recognition phase. First, cells A are put together with cells B. Then, cells A are “learning” to recognize cells B. If afterwards they are put in a petri dish with cells B, they will emit GPF. Otherwise, they will stay dark.<br />
<br />
=== Final project===<br />
<br />
The chosen project is a modified version of Learning which was presented at the last meeting. The system proposed is modified as in its current state may be implemented with two switches only, something which is not exciting enough. Keeping the idea of learning, and of training and testing phases, we have come up with educatETH <i>E.coli</i>.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/LabETHZ/Biology/Lab2007-10-20T08:22:47Z<p>Kdikaiou: Fixed headings, added references.</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|900px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - In the lab ::. </b></font></center><br><br />
<br />
In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section [https://2007.igem.org/ETHZ/Biology/Lab#.::_Problems_we_faced_::. Problems we faced] might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Biology | Biology Pespective]]. Finally, photos of our lab experience are accessible under [[ETHZ/Pictures | Pictures!]]<br />
<br />
Todo: decide what happens with lab book ([https://2007.igem.org/ETHZ/Lab_book here])<br />
<br />
==Cloning plan==<br />
<br />
===Parts assignment into plasmids===<br />
<br />
Three plasmids are used for the EducatETH <i>E.coli</i> system parts as follows:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Plasmids and contents'''<br />
|-<br />
! plasmid !! resistance !! copy type!! contents !! comments<br />
|-<br />
<br />
| [[ETHZ/pbr322| pbr322]] || ampicillin || high || 1,2,3 || constitutive subsystem<br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || chloramphenicol|| low || 4,5,8,9 || reporting subsystem<br />
|-<br />
<br />
| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| low || 6,7,10,11 || learning subsystem, reporting subsystem<br />
|-<br />
|}<br />
<br />
It is important to insert parts responsible for the production of fluorescent proteins in low copy plasmids, as they are potentially harmful for the cell. Unfortunately, working with low copy plasmids makes the procedure more demanding in the lab.<br />
<br />
===Linkers===<br />
<br />
Because the plasmids used were not standard plasmids found in the registry, but came from the lab where we work, linkers compatible with the standard BioBrick assembly have to be used in order to work with them. The list of all linkers is the following:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Linkers for plasmids'''<br />
|-<br />
! Linker!! Plasmid<br />
|-<br />
<br />
| [[ETHZ/pbr322-1| pbr322-1]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-2| pbr322-2]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-3| pbr322-3]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-4| pbr322-4]]|| pbr322 <br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || pck01 <br />
|-<br />
| [[ETHZ/pCK01-2| pck01-2]] || pck01 <br />
|-<br />
<br />
| [[ETHZ/pacyc177-1| pacyc177-1]] || pacyc177<br />
|-<br />
| [[ETHZ/pacyc177-2| pacyc177-2]]|| pacyc177<br />
|-<br />
|}<br />
<br />
Note that four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.<br />
<br />
=== Procedure ===<br />
<br />
The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. [[Image:Assembly _process.png|thumb|300px|DNA assembly process ([1]) '''(Fig. 4)''']] Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted ''before'' the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the ''donor'' and the plasmid accepting the new part will be referred to as the ''acceptor''. Composite pars made of parts '''a''' and '''b''' are denoted '''a.b'''.<br />
<br />
==== Plasmid 1 ''(pbr322ap)'' ====<br />
<br />
# Put parts 1,2,3 in pbr322ap plasmids. <br />
# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.<br />
# Merge plasmid containing part '''1''' ''(donor)'' with plasmid containing composite part '''2.3''' ''(acceptor)''. You should get a plasmid containing a '''1.2.3''' composite part.<br />
<br />
==== Plasmid 2 ''(pck01cm)''====<br />
<br />
# Put parts 4,5,8,9 in pck01cm plasmids. <br />
# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.<br />
# Merge plasmid containing part '''8''' ''(donor)'' with plasmid containing part '''9''' ''(acceptor)''. You should get a plasmid containing a '''8.9''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''4.5''' ''(donor)'' with plasmid containing composite part '''8.9''' ''(acceptor)''. You should get a plasmid containing a '''4.5.8.9''' composite part.<br />
<br />
====Plasmid 3 ''(pacyc177km)''====<br />
<br />
# Put parts 6,7,10,11 in pacyc177km plasmids. <br />
# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.<br />
# Merge plasmid containing part '''10''' ''(donor)'' with plasmid containing part '''11''' ''(acceptor)''. You should get a plasmid containing a '''10.11''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''6.7''' ''(donor)'' with plasmid containing composite part '''10.11''' ''(acceptor)''. You should get a plasmid containing a '''6.7.10.11''' composite part.<br />
<br />
== Problems we faced==<br />
<br />
== Lab book ==<br />
<br />
==== Week 1 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 06. Aug. 2007 <br />
|<br />
| <br />
* Preparing the Solutions <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Tue, 07. Aug. 2007 <br />
| <br />
|<br />
* Prepare competent cells for all parts<br />
* Transformation of all the parts <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Wed, 08. Aug. 2007 <br />
| <br />
| <br />
* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures) <br />
| Raphael<br>Stefan<br />
|-<br />
| Thu, 09. Aug. 2007 <br />
| <br />
| <br />
* MINIPREP of the grown (10) o/n cultures<br />
* Gelelectrophoresis of the grown cultures (step: 0.8% Agarose) <br />
| Raphael<br>Stefan<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Fri, 10. Aug. 2007 <br />
| <br />
| <br />
* 7 working parts/plasmids (step after "DIGESTS"): <br> (B0034, R0062, R0053, E0434, B0015, R0010, E0422)<br />
* 4 parts/plasmids minipreped: <br> (R0040, R0051, Q04121, C0053)<br />
|<br />
Christos <br> Markus <br> Stefan<br />
|-<br />
| Sat, 11. Aug. 2007 <br />
| <br />
| no labwork <br />
|<br />
|-<br />
| Sun, 12. Aug. 2007 <br />
|<br />
| labwork cancelled<br />
|<br />
|}<br />
<br />
==== Week 2 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 13. Aug. 2007 <br> start at 3 pm <br />
|<br />
* Prepare competent cells<br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053<br />
| <br />
* o/n culture (E.Coli Top10)<br />
* Control Restrictions (didn't work)<br />
| Martin<br> Markus <br> Christos <br> Tim <br><br />
|-<br />
| Tue, 14. Aug. 2007 <br />
| Morning Shift: <br><br />
* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br> <br />
Evening Shift: <br><br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
| Morning Shift: <br><br />
* Prepared competent cells (stored in the -80°C freezer in the basement) <br><br />
Evening Shift: <br><br />
* Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday) <br />
* Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4% <br />
|Morning Shift (9am-1pm?): <br> Markus <br> Tim <br> Evening Shift (5pm-...):<br> Martin <br> Christos<br />
|-<br />
| Wed, 15. Aug. 2007 <br />
| <br />
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434 <br />
|<br />
* Ligation didn't work due to bad quality of enzymes (probably) <br />
| From 12:<br> Martin<br>Markus<br><br><br />
|-<br />
| Thu, 16. Aug. 2007 <br />
| <br />
* Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)<br />
* Transformation of #13 and #14<br />
| <br />
* Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510) <br> One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)<br />
* Transformation of #13 (Q04121) and #14 (C0053) <br> Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)<br />
|<br />
Markus<br>Christos<br>(Martin)<br />
|-<br />
| Fri, 17. Aug. 2007 <br />
|<br />
* o/n of #13 and #14<br />
* Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful<br />
| <br />
* #13 and #14 didn't grow<br />
* # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates<br />
* New white pipette tips prepared (autoclave)<br />
* New bottles of Liquid LB and LB Agar prepared (autoclave)<br />
| Martin<br />
|-<br />
| Sat, 18. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 19. Aug. 2007 <br />
|<br />
|<br />
|<br />
|}<br />
<br />
==== Week 3 ====<br />
<br />
Little rearrangements of the parts. Planning of the sequences to order them.<br />
<br />
<br />
==== Week 4 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 27. Aug. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 28. Aug. 2007 <br />
| <br />
|<br />
| <br />
|-<br />
| Wed, 29. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Thu, 30. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 31. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 01. Sept. 2007 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 and plated them <br />
| Stefan<br />
|-<br />
| Sun, 02. Sept. 2007 <br />
|<br />
* Prepare o/n of pbr322, pcyc177, pck01<br />
| <br />
* o/n of pcyc177, pck01<br />
* the plates of pcyc177 and pck01 are in the fridge<br />
* transformed pbr322 because the culture didn't grow on the plate<br />
| Stefan<br />
|}<br />
<br />
==== Week 5 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 03. Sept. 2007 <br />
|<br />
* Prepare new competent cells<br />
* Miniprep pcyc177 and pcK01<br />
* prepare new o/n culture of pbr322<br />
* Run agarose gel of Minipreped plasmids<br />
| <br />
* New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)<br />
* Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)<br />
* pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday<br />
* new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids<br />
* 2 boxes of blue pipette tips are in the autoclave<br />
* Stefan ran the agarose gel (?) <br />
| Martin<br>Stefan<br />
|-<br />
| Tue, 04. Sept. 2007 <br />
|<br />
* Miniprep pcyc177 and pck01<br />
* cut the prepped plasmids to test if we've got the right ones<br />
* run agarose gel to test the cut and uncut ones <br />
* prepare new o/n of pbr322 (from Andy) <br />
|<br />
* Miniprep of pcyc177 and pck01 (but not yet tested)<br />
* Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep <br />
| Martin<br>Christian<br />
|-<br />
| Wed, 05. Sept. 2007 <br />
| <br />
* Miniprep of pbr322<br />
* Test-Digest of pcyc177 and pck01 and agarose gel...<br />
* Streak out all three plasmids on new plates, so we have them in reserve <br />
|<br />
* New Plate of pbr322.<br />
* Minipreps and Agarose Gels will be done tomorrow <br />
| Martin<br />
|-<br />
| Thu, 06. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01<br />
* Test with agarose gel <br />
| <br />
* Gel of the older plasmids -> plasmid present<br />
| Christian<br />
|-<br />
| Fri, 07. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01 <br />
| <br />
* Plasmids miniprepped<br />
| Martin<br />
|-<br />
| Sat, 08. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 09. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 6 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 10. Sept. 2007 <br />
| <br />
* Miniprep pBR322<br />
* annealing of different MCSs<br />
* Digest of pCK01 with BamHI+AseI<br />
* digest of pACYC177 with BamHI+PstI<br />
* digest of pBR322 with EcoRI+PstI <br />
all digests o/n<br />
| <br />
<br />
Christian<br />
| | <br />
|-<br />
| Tue, 11. Sept. 2007 <br />
|<br />
* Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work<br />
* 1x ligation of MCS inside backbones o/d, Trafo<br />
* 1x ligation of MCS inside backbones o/n<br />
| <br />
* plate all 3 plasmids for new minipreps<br />
|<br />
Christian<br />
|-<br />
| Wed, 12. Sept. 2007 <br />
|<br />
* Trafo of o/n ligations<br />
* o/n cultures of putative clones <br />
|<br />
|<br />
Christian<br />
|-<br />
| Thu, 13. Sept. 2007 <br />
| <br />
* Minipreps of putative clones pCK01-MCS and pBR322-MCS<br />
* control digests of putative clones<br />
* new o/n cultures of the putative clones of o/n ligations<br />
| <br />
| <br />
Christian<br />
|-<br />
| Fri, 14. Sept. 2007 <br />
| <br />
*separation of control digests of putative clones <br />
|<br />
'''*pBR322-MCS (Tet-selection) clone2 positive''' <br />
|<br />
Christian<br />
|-<br />
| Sat, 15. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 16. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 7 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 17. Sept. 2007 <br />
|<br />
* new digest of pACYC177 with BamHI+PstI o/n<br />
* digest of pACYC177, pBR322 AP<br />
* ligation of 177 and 322AP<br />
| <br />
* digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)<br />
* ligation of pBR322 o/n<br />
* 100 ml o/n culture to MAXIprep pacyc177<br />
* Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)<br />
| <br />
Christian <br> Martin, Raphael<br />
|-<br />
| Tue, 18. Sept. 2007 <br />
|<br />
* different control digests of pBR322-MCS (Tet) (see last week)<br />
* separation of pACYC177 digest<br />
* Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)<br />
* Transformation of the ligated pbr322 AP (MCS)<br />
* Prep pacyc177<br />
* Digest prepped pacyc177<br />
|<br />
> no DNA on pACYC177 digest-gel, only degradation smear<br> <br><br />
<br />
* Plates of pbr322 AP grew<br />
* No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)<br />
* Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )<br />
* Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.<br />
* Digest of pacyc177 with PstI o/n (pray that it will work!)<br />
* New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)<br />
* test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests<br />
| <br />
<br />
Christian <br><br />
Martin<br>Raphael<br />
|-<br />
| Wed, 19. Sept. 2007 <br />
| <br />
* o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated<br />
* Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)<br />
* check the digests of pacyc177 (pst) and pck01 (notI)<br />
* design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01 <br />
|<br />
|<br />
|-<br />
| Thu, 20. Sept. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 21. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 22. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 23. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 8 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 24. Sept. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 25. Sept. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 26. Sept. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 27. Sept. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 28. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 29. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 30. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
====Week 9 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 1. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 2. Oct. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 3. Oct. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 4. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 5. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 6. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 7. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==== Week 10 ====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 8. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 9. Oct. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 10. Oct. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 11. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 12. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 13. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 14. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==References==<br />
<p>[1] [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly Process]</p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/LabETHZ/Biology/Lab2007-10-20T08:19:07Z<p>Kdikaiou: Added references, removed parts (are shown in biology page)</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|900px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - In the lab ::. </b></font></center><br><br />
<br />
In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section [https://2007.igem.org/ETHZ/Biology/Lab#.::_Problems_we_faced_::. Problems we faced] might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Biology | Biology Pespective]]. Finally, photos of our lab experience are accessible under [[ETHZ/Pictures | Pictures!]]<br />
<br />
Todo: decide what happens with lab book ([https://2007.igem.org/ETHZ/Lab_book here])<br />
<br />
=='''.:: Cloning plan::.'''==<br />
<br />
==='''.:: Parts assignment into plasmids::.'''===<br />
<br />
Three plasmids are used for the EducatETH <i>E.coli</i> system parts as follows:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Plasmids and contents'''<br />
|-<br />
! plasmid !! resistance !! copy type!! contents !! comments<br />
|-<br />
<br />
| [[ETHZ/pbr322| pbr322]] || ampicillin || high || 1,2,3 || constitutive subsystem<br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || chloramphenicol|| low || 4,5,8,9 || reporting subsystem<br />
|-<br />
<br />
| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| low || 6,7,10,11 || learning subsystem, reporting subsystem<br />
|-<br />
|}<br />
<br />
It is important to insert parts responsible for the production of fluorescent proteins in low copy plasmids, as they are potentially harmful for the cell. Unfortunately, working with low copy plasmids makes the procedure more demanding in the lab.<br />
<br />
==='''.:: Linkers::.'''===<br />
<br />
Because the plasmids used were not standard plasmids found in the registry, but came from the lab where we work, linkers compatible with the standard BioBrick assembly have to be used in order to work with them. The list of all linkers is the following:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Linkers for plasmids'''<br />
|-<br />
! Linker!! Plasmid<br />
|-<br />
<br />
| [[ETHZ/pbr322-1| pbr322-1]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-2| pbr322-2]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-3| pbr322-3]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-4| pbr322-4]]|| pbr322 <br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || pck01 <br />
|-<br />
| [[ETHZ/pCK01-2| pck01-2]] || pck01 <br />
|-<br />
<br />
| [[ETHZ/pacyc177-1| pacyc177-1]] || pacyc177<br />
|-<br />
| [[ETHZ/pacyc177-2| pacyc177-2]]|| pacyc177<br />
|-<br />
|}<br />
<br />
Note that four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.<br />
<br />
==='''.:: Procedure::.'''===<br />
<br />
The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. [[Image:Assembly _process.png|thumb|300px|DNA assembly process ([1]) '''(Fig. 4)''']] Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted ''before'' the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the ''donor'' and the plasmid accepting the new part will be referred to as the ''acceptor''. Composite pars made of parts '''a''' and '''b''' are denoted '''a.b'''.<br />
<br />
===== '''.::Plasmid 1 ''(pbr322ap)''::.''' =====<br />
<br />
# Put parts 1,2,3 in pbr322ap plasmids. <br />
# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.<br />
# Merge plasmid containing part '''1''' ''(donor)'' with plasmid containing composite part '''2.3''' ''(acceptor)''. You should get a plasmid containing a '''1.2.3''' composite part.<br />
<br />
===== '''.::Plasmid 2 ''(pck01cm)''::.''' =====<br />
<br />
# Put parts 4,5,8,9 in pck01cm plasmids. <br />
# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.<br />
# Merge plasmid containing part '''8''' ''(donor)'' with plasmid containing part '''9''' ''(acceptor)''. You should get a plasmid containing a '''8.9''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''4.5''' ''(donor)'' with plasmid containing composite part '''8.9''' ''(acceptor)''. You should get a plasmid containing a '''4.5.8.9''' composite part.<br />
<br />
===== '''.::Plasmid 3 ''(pacyc177km)''::.''' =====<br />
<br />
# Put parts 6,7,10,11 in pacyc177km plasmids. <br />
# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.<br />
# Merge plasmid containing part '''10''' ''(donor)'' with plasmid containing part '''11''' ''(acceptor)''. You should get a plasmid containing a '''10.11''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''6.7''' ''(donor)'' with plasmid containing composite part '''10.11''' ''(acceptor)''. You should get a plasmid containing a '''6.7.10.11''' composite part.<br />
<br />
=='''.:: Problems we faced ::.'''==<br />
<br />
=='''.:: Lab book ::.'''==<br />
<br />
===='''.:: Week 1 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 06. Aug. 2007 <br />
|<br />
| <br />
* Preparing the Solutions <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Tue, 07. Aug. 2007 <br />
| <br />
|<br />
* Prepare competent cells for all parts<br />
* Transformation of all the parts <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Wed, 08. Aug. 2007 <br />
| <br />
| <br />
* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures) <br />
| Raphael<br>Stefan<br />
|-<br />
| Thu, 09. Aug. 2007 <br />
| <br />
| <br />
* MINIPREP of the grown (10) o/n cultures<br />
* Gelelectrophoresis of the grown cultures (step: 0.8% Agarose) <br />
| Raphael<br>Stefan<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Fri, 10. Aug. 2007 <br />
| <br />
| <br />
* 7 working parts/plasmids (step after "DIGESTS"): <br> (B0034, R0062, R0053, E0434, B0015, R0010, E0422)<br />
* 4 parts/plasmids minipreped: <br> (R0040, R0051, Q04121, C0053)<br />
|<br />
Christos <br> Markus <br> Stefan<br />
|-<br />
| Sat, 11. Aug. 2007 <br />
| <br />
| no labwork <br />
|<br />
|-<br />
| Sun, 12. Aug. 2007 <br />
|<br />
| labwork cancelled<br />
|<br />
|}<br />
<br />
===='''.:: Week 2 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 13. Aug. 2007 <br> start at 3 pm <br />
|<br />
* Prepare competent cells<br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053<br />
| <br />
* o/n culture (E.Coli Top10)<br />
* Control Restrictions (didn't work)<br />
| Martin<br> Markus <br> Christos <br> Tim <br><br />
|-<br />
| Tue, 14. Aug. 2007 <br />
| Morning Shift: <br><br />
* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br> <br />
Evening Shift: <br><br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
| Morning Shift: <br><br />
* Prepared competent cells (stored in the -80°C freezer in the basement) <br><br />
Evening Shift: <br><br />
* Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday) <br />
* Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4% <br />
|Morning Shift (9am-1pm?): <br> Markus <br> Tim <br> Evening Shift (5pm-...):<br> Martin <br> Christos<br />
|-<br />
| Wed, 15. Aug. 2007 <br />
| <br />
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434 <br />
|<br />
* Ligation didn't work due to bad quality of enzymes (probably) <br />
| From 12:<br> Martin<br>Markus<br><br><br />
|-<br />
| Thu, 16. Aug. 2007 <br />
| <br />
* Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)<br />
* Transformation of #13 and #14<br />
| <br />
* Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510) <br> One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)<br />
* Transformation of #13 (Q04121) and #14 (C0053) <br> Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)<br />
|<br />
Markus<br>Christos<br>(Martin)<br />
|-<br />
| Fri, 17. Aug. 2007 <br />
|<br />
* o/n of #13 and #14<br />
* Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful<br />
| <br />
* #13 and #14 didn't grow<br />
* # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates<br />
* New white pipette tips prepared (autoclave)<br />
* New bottles of Liquid LB and LB Agar prepared (autoclave)<br />
| Martin<br />
|-<br />
| Sat, 18. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 19. Aug. 2007 <br />
|<br />
|<br />
|<br />
|}<br />
<br />
===='''.:: Week 3 ::.'''====<br />
<br />
Little rearrangements of the parts. Planning of the sequences to order them.<br />
<br />
<br />
===='''.:: Week 4 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 27. Aug. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 28. Aug. 2007 <br />
| <br />
|<br />
| <br />
|-<br />
| Wed, 29. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Thu, 30. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 31. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 01. Sept. 2007 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 and plated them <br />
| Stefan<br />
|-<br />
| Sun, 02. Sept. 2007 <br />
|<br />
* Prepare o/n of pbr322, pcyc177, pck01<br />
| <br />
* o/n of pcyc177, pck01<br />
* the plates of pcyc177 and pck01 are in the fridge<br />
* transformed pbr322 because the culture didn't grow on the plate<br />
| Stefan<br />
|}<br />
<br />
===='''.:: Week 5 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 03. Sept. 2007 <br />
|<br />
* Prepare new competent cells<br />
* Miniprep pcyc177 and pcK01<br />
* prepare new o/n culture of pbr322<br />
* Run agarose gel of Minipreped plasmids<br />
| <br />
* New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)<br />
* Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)<br />
* pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday<br />
* new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids<br />
* 2 boxes of blue pipette tips are in the autoclave<br />
* Stefan ran the agarose gel (?) <br />
| Martin<br>Stefan<br />
|-<br />
| Tue, 04. Sept. 2007 <br />
|<br />
* Miniprep pcyc177 and pck01<br />
* cut the prepped plasmids to test if we've got the right ones<br />
* run agarose gel to test the cut and uncut ones <br />
* prepare new o/n of pbr322 (from Andy) <br />
|<br />
* Miniprep of pcyc177 and pck01 (but not yet tested)<br />
* Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep <br />
| Martin<br>Christian<br />
|-<br />
| Wed, 05. Sept. 2007 <br />
| <br />
* Miniprep of pbr322<br />
* Test-Digest of pcyc177 and pck01 and agarose gel...<br />
* Streak out all three plasmids on new plates, so we have them in reserve <br />
|<br />
* New Plate of pbr322.<br />
* Minipreps and Agarose Gels will be done tomorrow <br />
| Martin<br />
|-<br />
| Thu, 06. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01<br />
* Test with agarose gel <br />
| <br />
* Gel of the older plasmids -> plasmid present<br />
| Christian<br />
|-<br />
| Fri, 07. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01 <br />
| <br />
* Plasmids miniprepped<br />
| Martin<br />
|-<br />
| Sat, 08. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 09. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 6 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 10. Sept. 2007 <br />
| <br />
* Miniprep pBR322<br />
* annealing of different MCSs<br />
* Digest of pCK01 with BamHI+AseI<br />
* digest of pACYC177 with BamHI+PstI<br />
* digest of pBR322 with EcoRI+PstI <br />
all digests o/n<br />
| <br />
<br />
Christian<br />
| | <br />
|-<br />
| Tue, 11. Sept. 2007 <br />
|<br />
* Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work<br />
* 1x ligation of MCS inside backbones o/d, Trafo<br />
* 1x ligation of MCS inside backbones o/n<br />
| <br />
* plate all 3 plasmids for new minipreps<br />
|<br />
Christian<br />
|-<br />
| Wed, 12. Sept. 2007 <br />
|<br />
* Trafo of o/n ligations<br />
* o/n cultures of putative clones <br />
|<br />
|<br />
Christian<br />
|-<br />
| Thu, 13. Sept. 2007 <br />
| <br />
* Minipreps of putative clones pCK01-MCS and pBR322-MCS<br />
* control digests of putative clones<br />
* new o/n cultures of the putative clones of o/n ligations<br />
| <br />
| <br />
Christian<br />
|-<br />
| Fri, 14. Sept. 2007 <br />
| <br />
*separation of control digests of putative clones <br />
|<br />
'''*pBR322-MCS (Tet-selection) clone2 positive''' <br />
|<br />
Christian<br />
|-<br />
| Sat, 15. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 16. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 7 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 17. Sept. 2007 <br />
|<br />
* new digest of pACYC177 with BamHI+PstI o/n<br />
* digest of pACYC177, pBR322 AP<br />
* ligation of 177 and 322AP<br />
| <br />
* digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)<br />
* ligation of pBR322 o/n<br />
* 100 ml o/n culture to MAXIprep pacyc177<br />
* Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)<br />
| <br />
Christian <br> Martin, Raphael<br />
|-<br />
| Tue, 18. Sept. 2007 <br />
|<br />
* different control digests of pBR322-MCS (Tet) (see last week)<br />
* separation of pACYC177 digest<br />
* Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)<br />
* Transformation of the ligated pbr322 AP (MCS)<br />
* Prep pacyc177<br />
* Digest prepped pacyc177<br />
|<br />
> no DNA on pACYC177 digest-gel, only degradation smear<br> <br><br />
<br />
* Plates of pbr322 AP grew<br />
* No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)<br />
* Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )<br />
* Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.<br />
* Digest of pacyc177 with PstI o/n (pray that it will work!)<br />
* New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)<br />
* test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests<br />
| <br />
<br />
Christian <br><br />
Martin<br>Raphael<br />
|-<br />
| Wed, 19. Sept. 2007 <br />
| <br />
* o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated<br />
* Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)<br />
* check the digests of pacyc177 (pst) and pck01 (notI)<br />
* design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01 <br />
|<br />
|<br />
|-<br />
| Thu, 20. Sept. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 21. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 22. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 23. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 8 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 24. Sept. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 25. Sept. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 26. Sept. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 27. Sept. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 28. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 29. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 30. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 9 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 1. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 2. Oct. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 3. Oct. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 4. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 5. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 6. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 7. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 10 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 8. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 9. Oct. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 10. Oct. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 11. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 12. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 13. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 14. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
==References==<br />
<p>[1] [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly Process]</p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-20T08:17:12Z<p>Kdikaiou: Fixed headings, removed old Stefan's text.</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing educatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab In the Lab], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested on how educatETH ''E.coli'' was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
== Introduction ==<br />
<br />
<p> educatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
== The complete system ==<br />
<br />
<p>The biological design of EducatETH <i>E.coli</i> is presented on [[Image:new_learning_system3.png|thumb|left|300px|'''Fig. 1: '''educatETH ''E.coli'' System]] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=== Constitutive subsystem ===<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=== Learning subsystem ===<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=== Reporting subsystem ===<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
== System phases ==<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), with AHL present. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical and reports with the same XFP as in the training phase (YFP for aTc, CFP for IPTG) or if it recognizes a different chemical and reports with a different XFP (GFP for trained with aTc and recognizing IPTG, RFP for trained with IPTG and recognizing aTc).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! <br />
! aTc<br />
! IPTG<br />
! AHL<br />
! p22cII<br />
! cI<br />
! Reporting <br />
|- <br />
| '''Start'''<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| '''Learning'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| '''Seeing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Memorizing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Testing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| yes<br />
| no<br />
| gfp<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| yes<br />
| no<br />
| yes<br />
| rfp<br />
|- <br />
<br />
|}<br />
<br />
=== Further thoughts on the system phases ===<br />
<br />
TODO: put Stefan's new text here. Text should be small.<br />
<br />
<br />
== System parts ==<br />
<br />
EducatETH <i>E.coli</i> consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!) Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System Parts'''<br />
|-<br />
|-<br />
!width="111"| 1<br />
|width="285"| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
|width="250"| constitutive subsystem<br />
| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Composite System Parts'''<br />
|-<br />
|-<br />
! 1+2+3<br />
|width="285"| tetR + lacI + luxR production <br />
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]<br />
|width="250"| constitutive subsystem<br />
|width="157"| <partinfo>BBa_I739013 SpecifiedComponents</partinfo><br />
|-<br />
! 4+5<br />
| P22 cII + EYFP production <br />
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo><br />
|-<br />
! 8+9<br />
| cI + ECFP production <br />
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo><br />
|-<br />
! (4+5)+(8+9)<br />
| (P22 cII + EYFP) + (cI + ECFP) production <br />
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo><br />
|-<br />
! 6+7<br />
| cI + P22 cII production <br />
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]<br />
| learning subsystem<br />
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo><br />
|-<br />
! 10+11<br />
| RFP + GFP production <br />
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo><br />
|-<br />
! (6+7)+(10+11)<br />
| (cI + P22 cII) + (RFP + GFP) production <br />
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]<br />
| learning/reporting subsystem<br />
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo><br />
|}<br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
!width="111"| 1'<br />
| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
|width="250"| reporting subsystem<br />
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2'<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3'<br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4'<br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5'<br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6'<br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
!width="111"| 1"<br />
|width="285"| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
| proof of concept, no part of the system <br />
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2"<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|-<br />
! 3"<br />
| PoC composite<br />
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
== References ==<br />
<br />
<br />
== To Do ==<br />
<br />
=== New ===<br />
<p><ul><br />
<li> Update and correct parts in parts list. Write better in a table<br />
<li> Update and correct full system scheme<br />
<li> Update graph scheme (made by Stefan) using aTc, IPTG instead of it1,2 and ia1,2<br />
<li> Which reporters are active when? I think CFP and YFP are not active only during training. Change text if needed.<br />
<li> Proposed terminology: seeing, memorizing<br />
<li> What are GFP, RFP controlled by? Is the full system scheme correct there?<br />
<li> What are the “double promoters” mentioned?<br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> How was Sven’s standard notation on how to write differently proteins, dna, rna?<br />
<li> Fill in table completely, make it more reading-friendly<br />
<li> Make "In the lab page", replace links.<br />
<li> Put image with 11 system parts (updated one, created by katerina)<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/File:Katerina_Dikaiou.jpgFile:Katerina Dikaiou.jpg2007-10-20T08:03:20Z<p>Kdikaiou: </p>
<hr />
<div></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Meet_the_teamETHZ/Meet the team2007-10-20T07:54:31Z<p>Kdikaiou: /* Meet the ETH Zurich 07 team */ -added katerina stuff</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
__NOTOC__<br />
<br />
=Meet the ETH Zurich 07 team=<br />
<br />
In this page, you can find more information on this year's team of ETH Zurich. If you also want to see photos taken during the preparation for this year's iGEM, don't forget to see [[ETHZ/Pictures | Pictures!]] as well.<br />
<br />
[[Image:ETHZ_Group_Photo2.jpg|left|thumb|The ETHZ iGEM2007 Team, posing!|420px]] [[Image:ETHZ_Group_Photo3.jpg|right|thumb|The ETHZ iGEM2007 Team, with its missing members!|420px]]<br />
<br />
<br />
==Team description==<br />
<br />
Our team is a combination of hard-working biologists and engineers! :)<br />
===Undergraduate students===<br />
*[[Image:Martin_Brutsche.jpg|left|40px]] Hi, my name is '''Martin''', and I'm a Master Student in Biomedical Engineering at ETH Zurich. Before that, I did my Diploma studies in Mechanical Engineering, at the University of Applied Sciences in Constance, Germany. I love sailing, snooker and holidays in Denmark. I joined iGEM to learn more about synthetic biology and labwork ([https://2007.igem.org/User:brutsche more info]).<br><br />
*[[Image:Katerina_Dikaiou.jpg|left|40px]]: Hi, I am '''Katerina'''! I'm one of the engineers in the group - I hold a Diploma in Electrical and Computer Engineering from the Aristotle University of Thessaloniki, Greece, and I am currently a Master student of Biomedical Engineering at ETH Zurich. I enjoy working at the interface between engineering and biology, and I also love music, literature and (very) long walks([https://2007.igem.org/User:kdikaiou more info]).<br><br />
*[[Image:Raphael_Gubeli.jpg|left|40px]] Hi, I'm '''Raphael''', Master Student of Biotechnology at ETH Zurich. So, that's why you can find me mainly in the lab (day and night!). Anyway, if there is time to see the daylight, you can find me on my bicycle in the city, or skiing down the mountains. I joined iGEM because I think that there is really a need to find international standards like the "biobricks". Of course, despite having a lot of work, it is a lot of pleasure and fun, working on this project ([https://2007.igem.org/User:Raphael more info]).<br><br />
*[[Image:Foto_iGem_hoehnels.png|left|38px]] Hello! I'm '''Sylke Hoehnel''', B.Sc.-Biotech Student at the ETH Zurich. I was born in Germany, and have lived in London for four years. So, as you can see, I like jetting around the world. I found iGEM as a great opportunity to be part of a team, working on a fun project that's also related to my studies. Apart from Bio-books, I like art, swimming and snowboarding ([https://2007.igem.org/User:hoehnels more info]).<br><br />
*[[Image:Nan_Li.jpg|left|40px]] Hello, I am '''Nan'''! I grew up in China and got my Bachelor degree in [http://en.wikipedia.org/wiki/Zhejiang_University Zhejiang University] located in the Heaven City - Hangzhou, Zhejiang, China. Right now I am doing my Master study in Biomedical Engineering at [http://www.ethz.ch ETH Zürich]. During my spare time, I enjoy the painting in silence. I like hiking with friends and traveling around. The only means to keep my life refreshing is to experience things new ([https://2007.igem.org/Nan_Li more info]).<br><br />
*Stefan Luzi: My name is '''Stefan Luzi''', M.Sc-Biotechnology student at ETH Zurich, and member of the ETH iGEM team 2007. During my spare time (yes, I have a life besides my studies) I like hiking, rowing or cycling, and can never reject a cold jug of beer ;-) ([https://2007.igem.org/User:Stefan more info]).<br />
<br />
===Graduate students===<br />
*[[Image:Christos_Bergeles.jpg|left|40px]] Hoi zaeme, I am '''Christos'''. I graduated with a Diploma in Electrical and Computer Engineering, from the National Technical University of Athens, Greece, and currently I am a PhD Student at ETH Zurich, working with nice, little, autonomous, magnetic, wireless, reconfigurable, fluorescent, swallowable, biocompatible super geeky microrobots. I sleep in my lab currently, but all is good! ([http://christos.bergeles.net more info]).<br />
*[[Image:Tim_Hohm.jpg|left|35px]] Hello everyone, my name is '''Tim Hohm'''. I received a diploma in computer science in 2003, from the University of Dortmund, Germany. After staying for two years in the research institute caesar (in Bonn, Germany), 2006 I joined the Systems Optimization Group at ETH Zurich headed by Prof. Zitzler. My research focuses on the application of bio-inspired optimization techniques on systems biology problems ([http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm]).<br />
*Christian Kemmer: ([http://fm-eth.ethz.ch/eth/peoplefinder/FMPro?-db=phonebook.fp5&-format=pf%5fdetail%5fde.html&-lay=html&-op=cn&Typ=Staff&Suche%5fText=kemmer&Suche%5fText%5fpre=kemmer&-recid=3772770936&-find=/ more info])<br />
*[[Image:Joe_Knight.jpg|left|40px]] '''Joe Knight''' here. I am a PhD candidate performing research in biomedical engineering at the ETH. Before coming to the ETH I was the 2005-06 Grube Fellow at Stanford University's Biodesign Innovation Program. I joined iGEM because I believe some of the greatest innovations in biotechnology of tomorrow will be made in synthetic biology, and I hope to be part of it all! ([https://2007.igem.org/User:JoeKnight more info])<br />
*Markus Uhr: ([https://2007.igem.org/User:uhrm more info]).<br />
*[[Image:Rico_Mockel.jpg|left|40px]] Hi, I am '''Rico'''. I am a Ph.D student of the ETH Zurich, Switzerland. I am working at the Institute of Neuroinformatics. I received a diploma (equivalent to MSc) in Electrical Engineering from the University of Rostock, Germany. I did my diploma thesis at the Institute of Neuroinformatics in Switzerland where I developed a STDP floating-gate synapse in CMOS VLSI ([http://www.ricomoeckel.de more info]).<br />
<br />
===Advisors===<br />
*[[Image:Sven_Panke.jpg|left|thumb|Sven Panke|80px]]<br />
*Joerg Stelling: (homepage)</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZETHZ2007-10-18T14:18:39Z<p>Kdikaiou: Changed learning, memory, recognition.</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<br />
__NOTOC__<br />
<br />
<center><font size = '+2'><b> .:: ETH Zurich - educatETH ''E.coli'' ::. </b></font></center><br><br />
<br />
==='''.:: Introduction ::.'''===<br />
<p> It is the '''3<sup>rd</sup>''' time the [http://www.ethz.ch ETH Zurich] takes part in [https://2007.igem.org/wiki/index.cgi International Genetically Engineered Machine Competition] (iGEM). This year, our combined team of biologists and engineers has undertaken the task of educating <i>E.coli</i> ! [[Image:Educateth_Ecoli.png|thumb|'''Fig. 1:''' The ETH Zurich team undertook the task of engineering a learning mechanism for <i>E.coli</i>.|500px]]More specifically, in our project (Fig. 1), we are trying to create <i>E.coli</i> which have the ability to distinguish between two chemicals they may be exposed to after they have undergone a training phase.</p><br />
<br />
<p>Learning plays a major role for living organisms, as it enables them to survive by adapting to an ever-changing environment. Engineering a simple biological system which exhibits learning behavior is of great interest, as it can support our understanding of this procedure by comparison with natural systems. On the other hand, learning and memorizing plays an equally important role in engineering; from handwriting recognition on PDAs to plain logical circuits storing their binary state on computers, it can be found on numerous everyday life applications. Constructing a biological analogue of a simple memory as known from logic design can hopefully function as a biological building block from which more complex systems may be constructed. We therefore think that educatETH <i>E.coli</i> operates on an exciting interface between engineering and biology.</p><br />
<br />
<p>Stay in this page for an overview of how educatETH <i>E.coli</i> works, the motivation behind it and its possible future applications. If you want to see the biological design of our system and the parts that it consists of, or if you are interested in building it yourself and want to read the lab protocols, the [[ETHZ/Biology | Biology Perspective]] will be of interest to you. If you want to know more on how educatETH <i>E.coli</i> has been modeled and simulated, or on its equivalences to systems such as flip-flops and finite state machines, please visit the [[ETHZ/Engineering | Engineering Perspective]]. You may also want to visit [[ETHZ/Meet_the_team | Meet the Team]] for information regarding the team and [[ETHZ/Pictures | Pictures!]] for our photo gallery. Finally, in [[ETHZ/Internal | Team Notes]] you can read the notes exchanged by the team during preparation for the competition.</p><br />
<br />
====='''.:: Team Members ::.'''=====<br />
KICK PEOPLE TO HAVE A PROPER LINK<br />
<p>As [https://2007.igem.org/wiki/index.cgi iGEM] is a synthetic biology competition, the ETH Zurich team consists of balanced numbers of biology and engineering students. Our team members are: </p><ul> <br />
<li><i>Project advisors</i>: [https://2007.igem.org/User:sven Sven Panke], Joerg Stelling<br />
<li><i>Undergraduate students</i>: [https://2007.igem.org/User:brutsche Martin Brutsche], [https://2007.igem.org/User:kdikaiou Katerina Dikaiou], [https://2007.igem.org/User:Raphael Raphael Guebeli], [https://2007.igem.org/User:hoehnels Sylke Hoehnel], [https://2007.igem.org/Nan_Li Nan Li], [https://2007.igem.org/User:Stefan Stefan Luzi]<br />
<li><i>Graduate students</i>: [http://christos.bergeles.net Christos Bergeles], [http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm], [http://fm-eth.ethz.ch/eth/peoplefinder/FMPro?-db=phonebook.fp5&-format=pf%5fdetail%5fde.html&-lay=html&-op=cn&Typ=Staff&Suche%5fText=kemmer&Suche%5fText%5fpre=kemmer&-recid=3772770936&-find=/ Christian Kemmer], [https://2007.igem.org/User:JoeKnight Joseph Knight], [https://2007.igem.org/User:uhrm Markus Uhr], [http://www.ricomoeckel.de Rico Möckel]<br />
</ul><br />
<p>For more information on the team members, follow the links or visit [[ETHZ/Meet_the_team | Meet the Team]].</p><br />
<br />
<br />
====='''.:: <i>E.coli</i> Intelligence (E.I) - Specifications and analogies ::.'''=====<br />
<br />
<p>How can we make <i>E.coli</i> bacteria able to report us about their environment? Can we teach bacteria to tell if they have seen before a specific chemical? [[Image:System_blended4.png|thumb|350px| '''Fig. 2: '''educatETH ''E.coli'' System ]]</p><br />
<br />
<p>This problem is essentially broken down to constructing a toggle switch which can maintain the state it acquired during a training phase. In Logic Design, this is done using a JK flip-flop with a latch. With this approach, reporting in the testing phase may be implemented with AND gates using the state of the toggle and the current chemical as inputs.<br><br />
(Want to read more about this? Visit the [[ETHZ/Engineering| Engineering Perspective]]!)</p><br />
<br />
<p>Back to biology, a toggle switch has been successfully implemented in [1]. This toggle switch, however, changes states according to one input only as it has one operator site and therefore loses its previous state whenever the system is exposed to a different chemical. This means that it cannot memorize information. To overcome this, we modified the toggle switch in [1] using two operator sites. In this way, a second chemical acting as a “helper” substance present only in the training phase may be used so that the toggle maintains its state during testing.<br> <br />
(Want to see the biological design of our modified toggle? Visit the [[ETHZ/Biology | Biology Perspective]]!)<br><br><br></p><br />
<br />
====='''.:: A short system description ::.'''=====<br />
<br />
<p>EducatETH <i>E.coli</i> is able to recognize between two chemical substances (aTC and IPTG) it has previously been exposed to with the help of an external chemical signal (AHL).<br />
<br>In the first part of the training phase (''“learning”''), the system is exposed to one of the two chemicals (aTc and IPTG), causing a steady system behavior. In the second part of the training phase (''“memory”''), AHL, is added while all other chemicals are removed, allowing the system to maintain its state. Finally, in the testing phase (''“recognition”''), the system is exposed to any of the two chemicals again. Via comparison of the toggle's steady state with the system response the new chemical causes, the system recognizes if it has been exposed to this chemical before or not. <br />
<br>As seen in Table 1, four system states are possible in the testing phase. We chose to use two reporters to control which chemical educatETH ''E.coli'' is exposed to during the training phase (CFP for IPTG, YFP for aTc). When the system recognizes the same chemical substance in the "recognition phase" as in the "learning phase ", it will respond with the same reporter output as in the learning phase (YFP for aTc learned and recognized, CFP for IPTG learned and recognized). However, when the system recognizes a different chemical substance, than previously learned, it will respond with either a GFP (when aTc is learned and IPTG is recognized) or a RFP output (when IPTG is learned and aTc is recognized). Thus, the system response is determined uniquely. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! Training <br>chemical <br />
! Testing <br>chemical <br />
! Fluorescence <br>during training<br />
! Fluorescence <br> during testing <br />
|- <br />
| aTc<br />
| aTc<br />
| YFP<br />
| YFP<br />
|- <br />
| aTc <br />
| IPTG<br />
| YFP<br />
| GFP<br />
|- <br />
| IPTG <br />
| aTc <br />
| CFP<br />
| RFP<br />
|- <br />
| IPTG<br />
| IPTG <br />
| CFP<br />
| CFP<br />
|}<br />
<br />
====='''.:: References ::.'''=====<br />
[1]: Toggle Switch<br />
<br />
====='''.:: Acknowledgements ::.'''=====<br />
<ul><br />
<li><p>[http://europa.eu The European Union]</p><br />
<li><p>[http://www.ethz.ch The ETH Zurich]</p><br />
<li><p>[http://www.geneart.com GENEART]</p><br />
</ul><br><br />
====='''.:: Links ::.'''=====<br />
<ul><br />
<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]</p><br />
<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]</p></ul><br><br />
<br />
=='''.:: To Do ::.'''==<br />
====='''.:: New ones ::.'''=====<br />
<p><ul><br />
<li> How about changing "educatETH <i>E.coli</i> to all italics "<i>educatETH E.coli</i>"<br />
<li> Change the name of p22cII to only cII (its a cI analog) p22 just means that it derives from the phage p22.<br />
<li> IMPORTANT: THE COLOURS ARE TOTALLY WRONG IN THE SIMULATION PART. Please refer to the table on the main page. Changed it just now, it was wrong before!!!<br />
<li> Change banner on top of page. Stefan: Can we please change the banner back to the old version?!? It was MUCH nicer.<br />
<li> Decide on headings type. I like larger headings more, I also like the horizontal line because it separates, put it only on Introduction for you to see how it is.<br />
<li> Possibly put table of contents. <br />
<li> Possibly take educatETH <i>E.coli</i> from Figure.<br />
<li> Change Figure 2.<br />
<li> <span style=color:blue;>Possibly change figure caption format from xxx (Fig. x) to Fig.x: xxx. Put second format on Figure 2 for all to check.</span><br />
<li> <span style=color:blue;>Fix table showing possible system states and reporters. Maybe with some color? --> No colour please. </span><br />
<li> Put cross-reference on table. Is there something like a description function for tables? An automatic "Tab. 1" as in "Fig. 1"?<br />
<li> <span style=color:blue;>Align table left</span><br />
<li> <span style=color:blue;>Add which reporters do what in paragraph.</span><br />
<li> Put (compressed!) team photo instead of Fig.1<br />
<li> <span style=color:blue;>Fix paragraph with system description, looks somehow bulky. Maybe italics or bold on important things?</span><br />
<li> Fix reference on toggle switch.<br />
<li> Possibly remove Introduction heading altogether, and just start with the text.<br />
</ul></p><br />
*These two lines are kinda confusing...<br />
**" Main Page / Biology Pespective / Engineering Perspective / Meet the Team / Team Notes / Pictures!"<br />
**"Main Page / System Modeling / Simulations / System Implementation / Lab Notes / Meet the Team / Team Notes / Pictures!<br />
*<span style=color:red;>'''BTW, can we label the solved problems to <span style=color:blue;>blue <span style=color:red;>?'''<br />
<br />
====='''.:: Old ones ::.'''=====<br />
<p><ul><br />
<li>Need picture: Bacteria red, Bacteria Green, two pictures showing different exposure to chemicals (''Sylke: see my presentation Sven will hold for me tonight (20.09) -> the FACS guy wants probes of our XFP expressing E.coli - the one who brings it to him can just put it under the microscope and take a few pictures for the wiki'')<br />
<li>Need picture: Einstein ecoli (''Sylke: does Stefan have the layer file?'')<br />
<li>Stupidity: All E. Colis are equal, but our E. Colis are more equal than the others :D<br />
<li>''Katerina'': 2. If we have a separate "Meet the team" page (which is good that we have), we have to make sure that all data about each one of us (short bio+photo) appear also on each one's user page-the same version would be the best, in my opinion.<br />
<li>''Katerina'': 3. Figure 2, bottom right part needs to be a bit larger/more clear, in my opinion, as it's important. (Christos: If you click, then it becomes larger. Will have the same at the bio part as well... Should I make it bigger anyway? I will change it, it is wrong anyway :D)<br />
</ul></p><br />
<br />
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<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2007.igem.org/ETHZ" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2007.igem.org/ETHZ" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onError="this.onError=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com'" /><br />
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<html><body><br />
<a><br />
<img src="http://www.easycounter.com/counter.php?stefanluzi"<br />
border="0" alt="Welcome to ETHz - iGEM07"></a><br />
</body></html></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/ModelETHZ/Model2007-10-18T13:06:48Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
__NOTOC__<br />
<br />
====.:: Modeling the educatETH <i>E. coli</i> System ::.====<br />
<br />
As it has been already discussed in the main page, we are interested in designing a system that is able to adapt to its environment. Our ideas were based on discussions about neural networks, and on how we can create a biological system that exhibits the behavior of learning, without having to resort to evolutionary processes. <br />
<br />
[[Image:ETHz_Flowdiagram.png|thumb|Fig. 1: Flow diagram. This figure shows the protocol with which the final system should be tested as well as the test results in form of the reported colors. There are tree phases the systems has to perform: (1) a training or learning phase in which the system learns an input and stores it in its memory, (2) a memory phase in which the system has to keep the content of its memory and finally (3) a recognition phase where the output of the system depends on the content of its memory as well as on the current input.|450px]]<br />
<br />
Learning can be considered as a switching of behavior, based on some external stimuli. It comes thus naturally, to work on existing ideas of toggle switches and finite state automatons. <br />
<br />
In our system, we are able to distinguish only between two chemicals. The proposed is only a minimal system that should be able to act as a proof of concept. By introducing the ability to have more final states, in an abstract manner, this correlates with the level of "intelligence" of the biological system. A protocol on how the system should react according to an input is shown in Fig. 1.<br />
<br />
The idea behind this protocol is that <br />
* The system will be able to learn one of two input signals - aTc or IPTG - during a learning phase if no input signal AHL is present. Depending on the input it will report by producing either green or yellow florescence. <br />
* Once the system has learned, the inputs - aTc or IPTG - can be removed and the system goes into a memory state in the presence of the "helper" substance AHL. In this state no output color is reported. Memorizing is guaranteed by removing the input chemicals. This results in a following successful recognition phase.<br />
* During the recognition phase, the inputs aTc or IPTG are (re-)inserted. The system reports by changing its color depending on the input and its current memory state. This is why the system has different florescent properties even in the presence of the same input. The recognition phase takes place in the presence of AHL to keep the memory enabled and avoid another learning phase. Since we would like to separate four different end states for our system, we had to use four fluorescent proteins to encode them.<br />
<br />
==== .:: Model Overview ::. ====<br />
<br />
One can start developing our system with a top-down approach. We start with the classical back box approach as shown in Fig. 2. <br />
<br />
[[Image:ETHZBlackbox.png|thumb|Fig. 2: Black box |280px]]<br />
<br />
Based on what was discussed in the previous section, the properties that our system has can be summarized as follows:<br />
* We need two inputs that should be learned/detected/adapted to,<br />
* We need one input to switch on the memory.<br />
* We need to alternate between at least three states. That is why we decided to use two state variables - cI and p22cII. -------- ARE YOU SURE ABOUT THAT?<br />
* We need four florescent signals for the outputs. One could also decide to take six output signals into account, to further distinguish the learning phase from the recognition phase. However, we restricted ourselves to 4 outputs to reduce the number of genes that are needed to implement the signals.<br />
<br />
[[Image:ETHZFullsystemmodel.png|left|thumb|Fig. 3: System overview. AHL, IPTG and aTc are passing the cell membrane where they build complexes with the sensor proteins LuxR, LacI and TetR. These sensor proteins and/or complexes are used to control memory formation and the production of the reporter proteins. The memory content is represented by the proteins cI and p22cII that repress the production of each other. YFP, RFP, CFP and GFP stand for yellow, red, cyan and green florescent protein, respectively.|420px]]<br />
<br />
Based on the above, the internal structure of the system can be defined, and it can be seen in Fig. 3. However, we had to keep in mind that the proposed system should be implemented in DNA, and that it would be sensitive to noise. As a result, we took several actions to achieve better experimental results and easier DNA construction:<br />
* To be more robust against perturbations, we coupled the state variables cI and p22cII in the way that is well known from memory circuits. Based on this approach, one state variable is depressing the other one, and the system's internal toggle has the possibility of reaching two stable states.<br />
* Since - due to their size - proteins can only hardly pass the cell membrane (if they are not actively transported through the cell membrane), we decided to use the much smaller inducer molecules AHL, IPTG and aTc as inputs. However, since these inducers cannot directly act on the transcription of the DNA nor on the production of proteins, we need to produce the sensor proteins LuxR, LacI and TetR that build complexes with AHL, IPTG and aTc, respectively.<br />
* The sensor proteins and complexes are used to control the memory formation and the production of the florescent reporter proteins YFP, RFP, CFP and GFP.<br><br><br />
<br />
====.:: Detailed Model ::. ====<br />
In this section we are transferring our model into a more detailed descriptions of the involved molecules and proteins. <br />
<br />
==== Sensors ====<br />
<br />
As shown in Figure 4, the proteins that act as sensors for the inducer signals are constituitively produced. <br />
<br />
[[Image:Model01b.png|center|thumb|Fig. 4: The proteins that act as sensors are constituitively produced.|140px]]<br />
<br />
==== Memory ====<br />
<br />
The memory control are based on the following underlying mechanisms:<br />
* The sensor proteins form complexes together with the inducers. These complexes are used to either activate (in case of the complex consisting of luxR and AHL) or repress (in case of the complexes consisting of LacI and IPTG as well as TetR and aTc) the DNA transciption of the proteins cI and p22cII. <br />
* Futhermore, p22cII and cI repress the DNA transciption of each other.<br />
<br />
[[Image:ETHZModelLearning.png|center|thumb|Fig. 5: Learning system. Depending on the inputs IPTG or aTc the proteins cI and p22cII are produced.|300px]]<br />
<br />
* Figure 5 shows the protein production system that is used during the learning phase where there is still no cI or p22cII produced so far. If either IPTG or aTc is added, cI or p22cII are produced, respecively. Since no AHL is present the inner toggle switch (see Figure 6) is turned off.<br />
<br />
[[Image:ETHZModelMemory.png|center|thumb|Fig. 6: Memory system. If AHL is present the production of either cI or p22cII is continued.|420px]]<br />
<br />
* During the memory phase AHL is added and the IPTG and aTc are removed. That is why only the inner toggle switch (see Figure 6) is turned on while the protein production systems shown in Figure 5 are disactivated. Depending on what was produced during the learing phase either the production of cI or p22cII is continued. That is why the system can act as an memory that is actively keeping the information that it learned.<br />
<br />
The final assembly of the memory system is shown in Figure 7.<br />
<br />
[[Image:Model02b.png|center|thumb|Fig. 7: Final interaction of the learning and memory system. The memory content is represented by the concentrations of the proteins cI and p22cII.|560px]]<br />
<br />
==== Reporters ====<br />
<br />
Figure 8 gives an overview about the reporter system. Florescence reporter proteins are expressed depending on the inducer concentrations and the concentrations of cI and p22cII. E.g. both the presence of either TetR or cI will repress the production of YFP. However, if the inducer aTc is present aTc will bind to TetR which can no longer block the production of YFP.<br />
<br />
[[Image:Model03b.png|center|thumb|Fig. 8: The production of the florescence reporter proteins depends on the memory content (cI or p22cII) and the current input (aTc or IPTG).|600px]]<br />
<br />
== Equations ==<br />
<br />
To perform simulations we descibe our system with the help of simple differential equations. We use a simple notation. All concentrations are given in brackets like [cI]. All decay constants are described by a variable d followed by the name of the protein they refer to. We descibe the production of the proteins by a basic constant production level that models the leak of the production system and a factor of l and c<sub>max</sub> that descibe the maximum production of a protein given in [M]. Depending on if the DNA for a protein is implemented on a low or a high copy plasmid we distinguish between l<sub>lo</sub> and l<sub>hi</sub>, respectively.<br />
<br />
''For a more basic introduction into how to transfer our model into equations see the section [[ETHZ/Modeling_Basics|Modeling Basics]].''<br><br />
<br />
==== Constitutively produced proteins ====<br />
<br />
The equations for the constitutively produced proteins are very simple since we do not have to consider any dependence on other proteins. They are designed so that the protein concentration tends to l<sub>hi</sub>*c<sub>max</sub>/d for steady state.<br />
<br />
[[Image:Constitutive_braced.png|330px]]<br />
<br />
==== Allosteric regulation ====<br />
<br />
These equations descibe the formation of complexes between the inducers and sensor proteins. We do not use differential equations but by directly descibing the concentrations of the complexes which is a valid assumption if we alway wait until steady state.<br />
We descibe the total amount of proteins by the index 't' while we use the index '*' for proteins that built a complex with the respective inducer. E.g. <br />
* [TetR<sub>t</sub>] describes the total amount of TetR that is available while <br />
* [TetR<sub>*</sub>] describes the proteins that are available as a complex with aTc, and finally<br />
* [TetR] gives the concentration of free TetR proteins.<br />
<br />
[[Image:Eq04.png|208px]]<br />
<br />
==== Learning and memory system ====<br />
<br />
The learning and memory system is the most complicated part of our system due to the feedback between the state variables and proteins cI and p22cII as well as the variation of the production of these proteins depending on the inputs. The following equation descibe the concentrations of the memory proteins as a system of coupled differential equations. The equations consist of two major production parts and a decay part. <br />
* The first production part models the production of either cI or p22cII during the learning phase and corresponds to the model in Figure 5.<br />
* The second production part descibes the inner toggle switch that was shown in Figure 6.<br />
<br />
[[Image:Toggle_braced.png|770px]]<br />
<br />
==== Reporter system ====<br />
<br />
The equations for the reporter system finally descibe the production of the florescence proteins depending on the inputs and memory proteins as modeled in Figure 8. Note that both inputs and memory proteins act repressively on the production of the florescence proteins. So e.g. YFP is only produced when there is both no cI and all TetR is bind in a complex together with aTc.<br />
<br />
[[Image:Reporter_braced.png|778px]]<br />
<br />
''For information regarding how well our system performs have a look at the section [[ETHZ/Simulation|Simulations]].''<br><br />
''A more detailed overview about the parameters that we used to descibe our system is given in the section [[ETHZ/Parameters|Parameters]].''<br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/PicturesETHZ/Pictures2007-10-18T13:06:39Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
__NOTOC__<br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Pictures ::. </b></font></center><br><br />
<br />
=====.:: Brainstorming ::.=====<br />
<p><br />
[[Image:Brainstorming.jpg|left|thumb|300px|Brainstorming on the learning system]] [[Image:Brainstorming2.jpg|center|thumb|300px|Brainstorming on the learning system]]<br />
</p><br />
<br><br />
<br />
=====<b>.:: Working in the lab ::. </b><br>=====<br />
<p><br />
[[Image:Igem_bench.jpg|left|thumb|300px|Our iGEM bench!]] [[Image:Ecoli1.jpg|center|thumb|300px|Growing E. Coli]]<br />
[[Image:Ecoli2.jpg|left|thumb|300px|Growing more E. Coli!]]<br />
</p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/InternalETHZ/Internal2007-10-18T13:06:30Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+2'><b> .:: ETH Zurich - educatETH ''E.coli'' ::. </b></font></center><br><br />
<br />
=='''.:: Synthetic Biology Boot camp ::.'''==<br />
To make everybody familiar with synthetic biology concepts and to assist coming up with ideas for an interesting project, we will read and present publications on important topics. The list to be covered is the following: <br />
<br />
* Introduction to synthetic biology (Markus, Martin)<br />
* DNA de novo design (Rico, Raphael)<br />
* DNA circuits (Christian, Nan))<br />
* Hysteresis (Tim, Sylke)<br />
* Oscillators (Christos, Joe, Katerina and Markus, Martin)<br />
* Zinc fingers (Sylke)<br />
* Noise in single cell measurements<br />
* Distance communication (Rico, Raphael)<br />
* Parameter manipulations (Christos, Joe, Katerina)<br />
* Orthogonal systems (Christos, Joe, Katerina)<br />
* Minimal genomes (Markus, Martin)<br />
* RNA regulators (Rico, Raphael)<br />
<br />
All presentations have been uploaded in the known web space. You may also contact the people who did it if you need additional information.<br />
<br />
=='''.:: Brainstorming ::.'''==<br />
<br />
==='''.:: Brainstorming sessions::.'''===<br />
During the first two weeks of July, the team has to come up with a project. The team is divided into three groups, which will brainstorm individually. Sven and Joerg are available on the 4th and 5th of July in case groups needs to consult them. The objective is that each group comes up with many fancy ideas. To facilitate this, keep in mind the following "brainstorming rules":<br />
<br />
# Defer judgment - the rules of nature don't apply<br />
# Encourage wild ideas<br />
# Build on the ideas of others<br />
# Be visual<br />
# Go for quantity<br />
# Stay focused on topic<br />
<br />
==='''.:: Preliminary ideas ::.'''===<br />
<br />
* '''PID Controller''': Design a PID controller out of biological elements. The P component can be a simple output to a regulatory protein, and the I component can be the overall protein production at a time period. What can the D component be?<br />
* '''Motion Detector''': Cells are grown on a petri dish. Below the dish, moving images are displayed. A 3-state automaton is proposed. Output A is created when light is present. Output B is created when light is absent. Moving patterns will cause some cells to create both outputs over time. This will result in some “inspector” cells producing output C, by collecting outputs A and B.<br />
* '''Analog-to-Digital Converter''': Compare the level of protein concentration with thresholds, and digitize the output.<br />
* '''Neural Network''': Create a sort of biological neural network with bacteria. We should address the issue of learning, and find a way to incorporate the feedback in the cell decision making process. Directed evolution can be a sort of feedback, but we want to avoid this. (This idea was the basis for the “learning project”)<br />
* '''Paramedic Cells''': Some cells are able to detect signals coming from other cells, and create food for them, or create proteins in order to save them and make them function better.<br />
* '''Cell Batteries''': Cells are able to create and store large quantities of ATP, during a “storing process”. Afterwards, they can detect a signal and give back all the energy they stored, in a short burst, like a capacitor. Other ideas are that the cells can “blow up” and emit large amounts of GFP, based on the ATP that they have accumulated.<br />
* '''Flashing Bacteria''': Cells are grown on a light pattern. The cells that are on the bright parts of the image are oscillating in phase, while the others are remaining dark. This results in the observation of a flashing pattern.<br />
* '''Biocam''': Visible to Fluorescent light converter.<br />
* '''BioCD''': “Print” cells on a film, then read them out and “reconstruct” the original data. Basically, it is an analog to digital converter, followed by a system that can interpret the digitized data. (This idea was the basis for the “Music of life project”, where cells would produce fluorescent proteins based on an analog input. Then, the amount and type of fluorescence would code some music).<br />
* '''Clock''': A follow-the-leader system. We have to groups of cells. The first group creates something that repels the second group. The second group creates a protein that attracts the first group. This way, they first group wants to “catch” the second group, whereas the second group wants to “avoid” the first group. This results in them moving around. We can say that the second group is the leader, and the first group exhibits a "follow-the-leader” behavior.<br />
* '''Sensors''': Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.<br />
<br />
==='''.:: Preferred projects::.'''===<br />
Three ideas of the above are chosen for further examination. The team will be again split up in three groups (different than before, to make sure that new ideas come up with mxing of people). Each group has to come up with an initial system, with remarks on its feasibility and coolness. Our results<br />
will be presented to all team members, so that potential projects may be limited down to two and subsequently to<br />
one. The preferred projects are: <br />
<br />
# '''Music of Life''': The basic idea is that instead of having an analog-to-digital converter with four outputs (three fluorescent proteins, and no output), we can have two switches. When switch A is on, RFP is produced. When switch B is on, GFP is produced. When both switches A and B are on, a yellowish output is observed. By recording these outputs, we can later create music, by assigning each fluorescent protein to a chord. For example, RFP would correspond to a G chord. The strength of the fluorescence can signify the strength of the chord. If the cells are placed on a spinning disk, we can have something like a vinyl player. A camera is observing the cells, and music is created on the fly. <br />
# '''Learning''': Based on the idea of the neural network, we want to create a biological system, where the cells can learn a specific behavior. In order to simplify the system, we decided that the cells can learn to recognize a specific type of other cells. We divide the process in a learning phase, and a recognition phase. First, cells A are put together with cells B. Then, cells A are “learning” to recognize cells B. If afterwards they are put in a petri dish with cells B, they will emit GPF. Otherwise, they will stay dark.<br />
<br />
==='''.:: Final project::.'''===<br />
<br />
The chosen project is a modified version of Learning which was presented at the last meeting. The system proposed is modified as in its current state may be implemented with two switches only, something which is not exciting enough. Keeping the idea of learning, and of training and testing phases, we have come up with educatETH <i>E.coli</i>.<br />
<br />
=='''.:: Task List ::.'''==<br />
<br />
==='''.:: Project Task List ::.'''===<br />
<br />
The things to do, from the most pressing (timewise), to the least pressing (timewise) is below. Please put your name next to the task that you believe that you can undertake.<br />
<br />
# '''Team descriptions (overdue) and team photograph''' <br> ''Christian'': I guess I am the only semiprofessional photographer of the group. I can do some group pictures etc. but for this we need some ideas... - I would like to do something special. I also made the group-pics of the Synth. Biology 3.0 conference. Some references ;-) : [http://www.fotocommunity.de/pc/pc/mypics/461397] <br> ''Raphael'': What about a short movie of us?<br />
# '''Team rosters due (1.9.)'''<br> ''Martin'': Does somebody know, what exactly should be done here? <br> ''Nan'': A list of team members, including some basic personal info.? (e.g. nationality, background, pet peeves...? )<br>''Katerina'': Guys, to make this easier, either write things about you on your personal page on the wiki or link to a page about you. This way it'll be easy to put it all together afterwards.<br />
# '''Labwork (parts have to be at the registry in Boston on 26.10.)''' <br> ''Joe'': I can be in the lab at least 2 evenings a week and some times through the weekdays. <br> ''Martin'': From Monday I can work every day for the whole day. At the moment I only work for several hours... <br> ''Rico'': I have my exam on Tuesday. Afterwards I can assist.<br> ''Christian'': I can do the introduction of the polylinker into the vectors beside my normal labwork on the Hoenggerberg. I could also do the whole biobrick assembly if you want this (I will go on holiday from the 5.10-24.10.07) <br> ''Raphael'': That's the part where I will mainly contribute, from 14.09. on I can work several days/week<br> ''Christos'': I can assist at the afternoons, if needed. <br> ''Katerina'': 7.09 - 30.09 generally plenty of time, apart from when I do my semester project presentation, will keep you posted when that is. <br> ''Sylke'': I'll not be available until 14th of October but from then on I can work several days per week fulltime.<br />
# '''Testing/ Analysis/ Detection (due to 02.11.)''' <br> ''Sylke'': Meeting with Alfredo Franco-Obregon (FACS guy from center) on 18.09. concerning devices available for detection of GFP/ derivatives.<br />
# '''Simulation and sensitivity analysis'''<br> ''Martin'': From 10. Sept. I've got plenty time to work on it. I think Markus would join here too ;-) <br> ''Tim'': I can contribute in running stuff and help identify parameters from literature <br> ''Rico'': I can do simulations, sensitivity analysis. <br> ''Nan'': I would like to do simulations and sensitivity analysis. <br> ''Christos'': Yeap, I guess I can be here too. I will check some toolboxes to automate things, this weekend. <br> ''Katerina'': Want to help (parameter identification, programming, sensitivity analysis). Discuss in upcoming meeting tasks, versioning and ask Christian about parameters.<br />
# '''Presentation''' <br> ''Joe'': I'm American... I can sale anything.<br> ''Martin'': I'm bad in Layout stuff, but maybe I can help as an idea supplier or so. <br> ''Rico'': I like giving presentations. For preparation we will need a mixture of different background and excellent pictures!!! <br> ''Christian'': I can provide the molecular biology knowledge and part. <br> ''Nan'': I can work on the slides. <br> ''Christos'': I like this part, I guess everyone will contribute anyway...<br> ''Katerina'': I believe I can be of help in structure, layout and fancy stuff. Could help train a bit the people we decide to do the presentation (question answering, style). I also think that Joe and Christos could be good for doing the presentation (structured, pleasant voices and lively), can discuss this in a meeting. <br> ''Sylke'': I can help with the bio-part and I'm good at doing graphics and stuff. Can be a help when it needs to look good :-) <br />
#''' Poster''' <br> ''Joe'': See 5. above <br> ''Rico'': I can help. <br> ''Christian'': I can provide the molecular biology knowledge and part. <br> ''Nan'': Partly art. I will help. <br> ''Christos'': You can sell, but can you trick? lol :)<br> ''Katerina'': Not my strong point, but have an eye for typos, fonts, layout etc, so can help in final checking. <br> ''Sylke'': See above. Good at layout.<br />
# '''Wiki (Project and part documentation due on 26.10)''' <br> ''Martin'': See the points above, from next Monday I will give everything, now I'm doing my best... <br> ''Rico'': I can help. I guess this will have to contain the materials that we will also use for poster and presenttion anyway. <br> ''Nan'': Shouldn't it be updated with every going on process? <br> ''Christos'': I am trying to put stuff in, as it comes along. I will update the bio pages with the presentation material, this weekend (I hope). <br> ''Katerina'': You guys have done a great job so far, will help with whatever needed.<br />
# '''T-Shirts''' <br> ''Sylke'': in print (2007-09-24)</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Meet_the_teamETHZ/Meet the team2007-10-18T13:06:21Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
__NOTOC__<br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Meet the team::. </b></font></center><br><br />
<br />
In this page, you can find more information on this year's team of ETH Zurich. If you also want to see photos of ourselves and of the preparation for this year's iGEM, don't forget to see [[ETHZ/Pictures | Pictures!]] as well.<br />
<br />
====='''.:: To Do ::.'''=====<br />
<p><ul><br />
<li> Every one: put a small profile on your page.<br />
<li> Every one: send Christos a photo.<br />
<li> Decide what info will be put here, in which order etc.<br />
</ul></p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/EngineeringETHZ/Engineering2007-10-18T13:06:10Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
__NOTOC__<br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
=====.:: Introduction ::.=====<br />
<br />
<p><br />
Having decided to work on an engineered biological system which exhibits learning, we elaborated on its design. Discussing with the biologists of the team, we realized that what we knew from the field of logic design as JK flip-flop with a latch may be implemented with biological parts using a modified toggle switch. Initial simulations showed us that it was possible to reach the desired behaviour. Therefore, a complete framework of differential equations describing the system was constructed and parameters were searched in the literature. Simulations performed with our new detailed model are very encouraging.<br />
In this page, the equations that model our system are and explained. The values that were chosen for the system parameters are presented and the results of our simulations are analyzed. References are provided at the end of the page. For an introduction to system modeling in synthetic biology, please read our modeling tutorial [[ETHZ/Modeling_Basics|here]].</p><br><br />
<br />
=====.:: System Model ::.=====<br />
<br />
<p>Following the guidelines presented in our modeling guide, we divided the biological system into subsystems, each of which was modelled with a system of differential equations. According to what presented in the [[ETHZ/Biology|Biology Perspective]], our system is composed the following three subsystems:<br><br />
# A subsystem of constitutively produced proteins (see Fig. 1), <br />
# The learning subsystem(see Fig. 2), and <br />
# The reporting subsystem (see Fig. 3). <br />
The first two subsystems interact, and thus, they should be considered together. The third subsystem has no feedback with the other two, as it is only used for producing the appropriate fluorescent proteins.<br />
<br />
The subsystem of the constitutively produced proteins serves as a regulatory system, and can be modeled with the following three decoupled partial differential equations (see Fig. 1):<br />
<br />
[[Image:constitutive_part.png|center|thumb|'''Fig.1: '''Subsystem 1-Constitutively produced proteins|340px]]<br />
<br />
The second subsystem is the main part of our model. It stores the information concerning the learned information for the chemical of interest, and drives the production of the appropriate reporter during the recognition phase. It is actually a toggle switch that reaches its steady state depending on the chemical that it has been previously exposed to (see Fig. 2):<br />
<br />
[[Image:toggle_part_b.png|center|thumb|'''Fig.2: '''Subsystem 2-Basic learning subsystem (toggle)|900px]]<br />
<br />
The third subsystem reports the state that our system is, during the different phases of learning and recognition. During the learning phase, this subsystem reports which chemical the cells are exposed to, and during the recognition phase, it reports if the cells recognize the chemicals that they are currently exposed to (see Fig. 3):<br />
<br />
[[Image:reporting_part_b.png|center|thumb|'''Fig.3: '''Subsystem 3-Reporting subsystem |900px]] <br />
<br />
Note that the three constitutively produced proteins LacI, TetR and LuxR exist in two different forms; as free proteins and in complexes they build with IPTG, aTc and AHL, respectively. We need to model this complex-forming procedure, with another set of differential equations (Fig. 4):<br />
<br />
[[Image:allosteric_part.png|center|thumb|'''Fig.4: '''Allosteric regulation|240px]]<br />
<br />
In order to have meaningful results from our simulations, we browsed through the literature in order to find appropriate values for our parameters. We reduced our parameter space by joining parameters together, and we gave reasonable estimates, for the values that could not be extracted from available publications. Since this is a hard part that every team has to face, we present the table with the chosen parameters below:<br />
<br />
[[Image:parameters.png|center|thumb|'''Fig.5: '''Parameters of the model|800px]]<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Parameter <br />
! Value<br />
! Description<br />
! References<br />
|-<br />
| c<sub>1</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of<br> constitutive promoter <br>(per gene)<br />
| Estimate<br />
|-<br />
| c<sub>2</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of<br> luxR-activated promoter <br>(per gene)<br />
| Estimate<br />
|-<br />
| l<sup>hi</sup><br />
| 25<br />
| mid-copy plasmid number<br />
| Estimate<br />
|-<br />
| l<sup>lo</sup><br />
| 5<br />
| low-copy plasmid number<br />
| Estimate<br />
|-<br />
| a<sub>p22cII,LacI</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> p22cII/LacI-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>p22cII</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> p22cII-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>cI,TetR</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> cI/TetR-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>cI</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> cI-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>p22cII,TetR</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> p22cII/TetR-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>cI,LacI</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> cI/TetR-inhibited genes<br />
| Discussion<br />
|-<br />
| d<sub>LacI</sub><br />
| 2.31e-3 [pro sec]<br />
| degradation of lacI<br />
| [10]<br />
|-<br />
| d<sub>TetR</sub><br />
| 1e-5 [pro sec]/2.31e-3 [pro sec]<br />
| degradation of TetR<br />
| [9], [10]<br />
|-<br />
| d<sub>LuxR</sub><br />
| 1e-3 - 1e-4 [per sec]<br />
| degradation of LuxR<br />
| [6]<br />
|-<br />
| d<sub>cI</sub><br />
| 7e-4 [per sec]<br />
| degradation of cI<br />
| [7]<br />
|-<br />
| d<sub>p22cII</sub><br />
| <br />
| degradation of p22cII<br />
|-<br />
| d<sub>YFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of YFP<br />
| suppl. mat. to Ref. [8]<br />
|-<br />
| d<sub>GFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of GFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>RFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of RFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>CFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of CFP<br />
| in analogy to YFP<br />
|-<br />
| K<sub>LacI</sub><br />
| 1.3e-3 - 2e-3 [mM/h]<br />
| lacI repressor <br>dissociation constant<br />
| [2], [5]<br />
|-<br />
| K<sub>IPTG</sub><br />
| 1.5e-10 [mM/h]<br />
| IPTG-lacI repressor <br>dissociation constant<br />
| [5]<br />
|-<br />
| K<sub>TetR</sub><br />
| 5.6 (+-2) [nM-1]<br />
| tetR repressor <br>dissociation constant<br />
| [1]<br />
|-<br />
| K<sub>aTc</sub><br />
| 1120 (+-400) [nM-1]<br />
| aTc-tetR repressor <br>dissociation constant<br />
| [1], [3]<br />
|-<br />
| K<sub>LuxR</sub><br />
| <br />
* 0.003 [mM/s]<br />
* 55 - 520 [nM]<br />
| luxR activator <br>dissociation constant<br />
| [6]<br />
|-<br />
| K<sub>AHL</sub><br />
| <br />
* 0.009 [mM/s] - 0.1 [mM/s]<br />
* 0.09 - 1 [&#181;M]<br />
| AHL-luxR activator <br>dissociation constant<br />
| [6]<br />
|-<br />
| K<sub>cI</sub><br />
| 2e-3 [mM/h]<br />
| cI repressor <br>dissociation constant<br />
| [5]<br />
|-<br />
| K<sub>p22cII</sub><br />
| <br />
| p22cII repressor <br>dissociation constant<br />
|-<br />
| n<sub>LacI</sub><br />
| 1<br />
| lacI repressor <br>Hill cooperativity<br />
| [5]<br />
|-<br />
| n<sub>IPTG</sub><br />
| 2<br />
| IPTG-lacI repressor <br>Hill cooperativity<br />
| [5]<br />
|-<br />
| n<sub>TetR</sub><br />
| 3<br />
| tetR repressor <br>Hill cooperativity<br />
| [3]<br />
|-<br />
| n<sub>aTc</sub><br />
| 2 (1.5-2.5)<br />
| aTc-tetR repressor <br>Hill cooperativity<br />
| [3]<br />
|-<br />
| n<sub>LuxR</sub><br />
| 2<br />
| luxR activator <br>Hill cooperativity<br />
| [6]<br />
|-<br />
| n<sub>AHL</sub><br />
| 1<br />
| AHL-luxR activator <br>Hill cooperativity<br />
| [3]<br />
|-<br />
| n<sub>cI</sub><br />
| 1.9<br />
| cI repressor <br>Hill cooperativity<br />
| [5]<br />
|-<br />
| n<sub>p22cII</sub><br />
| <br />
| p22cII repressor <br>Hill cooperativity<br />
|-<br />
|}<br />
<br />
<br />
<br />
</p><br><br />
<br />
=====<b>.:: Simulations ::.</b><br>=====<br />
<br />
=====.:: References ::.=====<br />
<p><br />
[1] Weber W., Stelling J., Rimann M., Keller B., Daoud-El Baba M., Weber C.C., Aubel D., and Fussenegger M., <i>"A synthetic time-delay circuit in mammalian cells and mice"</i>, Proceedings of the National Academy of Sciences, vol. 104, no. 8, pp. 2643, 2007.<br><br />
[2] Setty Y., Mayo AE, Surette MG, and Alon U., <i>"Detailed map of a cis-regulatory input function"</i>, Proceeding of the National Academy of Sciences, vol. 100, no. 13, pp. 7702--7707, 2003.<br><br />
[3] Braun D., Basu S., and Weiss R., <i>"Parameter Estimation for Two Synthetic Gene Networks: A Case Study"</i>, IEEE Int'l Conf. Acoustics, Speech, and Signal Processing 2005, vol. 5, 2005.<br><br />
[4] Fung E., Wong W.W., Suen J.K., Bulter T., Lee S., and Liao J.C., <i>"A synthetic gene--metabolic oscillator"</i>, Nature, vol. 435, no. 7038, pp. 118--122, 2005, supplementary material.<br><br />
[5] Iadevaia S., and Mantzaris N.V., <i>"Genetic network driven control of PHBV copolymer composition"</i>, Journal of Biotechnology, vol. 122, no. 1, pp. 99--121, 2006.<br><br />
[6] Goryachev AB, Toh DJ, and Lee T., <i>"Systems analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constants"</i>, Biosystems, vol. 83, no. 2-3, pp. 178--187, 2006.<br><br />
[7] Arkin A., Ross J., and McAdams H.H., <i>"Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-Infected Escherichia coli cells"</i>, Genetics, vol. 149, no. 4, pp. 1633--1648, 1998.<br><br />
[8] Colman-Lerner A., Chin T.E., and Brent R., <i>"Yeast Cbk1 and Mob2 Activate Daughter-Specific Genetic Programs to Induce Asymmetric Cell Fates"</i>, Cell, vol. 107, no. 6, pp. 739--750, 2001.<br><br />
[9] Becskei A., and Serrano L., <i>"Engineering stability in gene networks by autoregulation"</i>, Nature, vol. 405, no.6786, pp.590--593, 2000.<br><br />
[10] Tuttle L.M., Salis H., Tomshine J., and Kaznessis Y.N., <i>"Model-Driven Designs of an Oscillating Gene Network"</i>, Biophysical Journal, vol. 89, no. 6, pp. 3873--3883, 2005.<br><br />
<br />
</p><br><br />
[http://www.pnas.org/cgi/content/abstract/104/8/2643 &#91;1&#93; Weber W et al.] <i>"A synthetic time-delay circuit in mammalian cells and mice"</i>, P Natl Acad Sci USA 104(8):2643-2648, 2007<br /><br />
[http://www.pnas.org/cgi/content/full/100/13/7702?ck=nck &#91;2&#93; Setty Y et al.] <i>"Detailed map of a cis-regulatory input function"</i>, P Natl Acad Sci USA 100(13):7702-7707, 2003<br /><br />
[http://ieeexplore.ieee.org/iel5/9711/30654/01416417.pdf &#91;3&#93; Braun D et al.] <i>"Parameter Estimation for Two Synthetic Gene Networks: A Case Study"</i>, ICASSP 5:769-772, 2005<br /><br />
[http://www.nature.com/nature/journal/v435/n7038/suppinfo/nature03508.html &#91;4&#93; Fung E et al.] <i>"A synthetic gene--metabolic oscillator"</i>, Nature 435:118-122, 2005 (supplementary material)<br /><br />
[http://dx.doi.org/10.1016/j.jbiotec.2005.08.030 &#91;5&#93; Iadevaia S and Mantzais NV] <i>"Genetic network driven control of PHBV copolymer composition"</i>, J Biotechnol 122(1):99-121, 2006<br /><br />
[http://dx.doi.org/10.1016/j.biosystems.2005.04.006 &#91;6&#93; Goryachev AB et al.] <i>"Systems analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constants"</i>, Biosystems 83(2-3):178-187, 2004<br /><br />
[http://www.genetics.org/cgi/content/abstract/149/4/1633 &#91;7&#93; Arkin A et al.] <i>"Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-Infected Escherichia coli cells"</i>, Genetics 149: 1633-1648, 1998<br /><br />
[http://download.cell.com/supplementarydata/cell/107/6/739/DC1/index.htm &#91;8&#93; Colman-Lerner A et al.] <i>"Yeast Cbk1 and Mob2 Activate Daughter-Specific Genetic Programs to Induce Asymmetric Cell Fates"</i>, Cell 107(6): 739-750, 2001 (supplementary material)<br /><br />
[http://www.nature.com/nature/journal/v405/n6786/abs/405590a0.html &#91;9&#93; Becskei A and Serrano L] <i>"Engineering stability in gene networks by autoregulation"</i>, Nature 405: 590-593, 2000<br /><br />
[http://www.biophysj.org/cgi/content/full/89/6/3873?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1&FIRSTINDEX=0&volume=89&firstpage=3873&resourcetype=HWCIT &#91;10&#93; Tuttle et al.] <i>"Model-Driven Designs of an Oscillating Gene Network"</i>, Biophys J 89(6):3873-3883, 2005<br /><br />
[http://www.pnas.org/cgi/reprint/99/2/679 &#91;11&#93; McMillen LM et al.] <i>"Synchronizing genetic relaxation oscillators by intercell signaling"</i>, P Natl Acad Sci USA 99(2):679-684, 2002<br /><br />
[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html &#91;12&#93; Basu S et al.] <i>"A synthetic multicellular system for programmed pattern formation"</i>, Nature 434:1130-1134, 2005<br /><br />
<br />
=====.:: To Do ::.=====<br />
<br />
*''Christos'': 1. Add gifs concerning the simulations<br />
*''Christos'': 2. Remove the table with the parameters, once we have satisfying values.<br />
*''Katerina'': 1. "The third subsystem has no feedback with the other two, as it is only used for producing the appropriate fluorescent proteins." Is this really true? gfp and rfp are situated in bigger parts with functionality. I think this needs to be rewritten more clearly.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-18T13:05:42Z<p>Kdikaiou: </p>
<hr />
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<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
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<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing educatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab In the Lab], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested on how educatETH ''E.coli'' was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
====.:: Introduction ::.====<br />
<br />
<p> educatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
====.:: The whole system ::.====<br />
<br />
<p>The biological design of educatETH <i>E.coli</i> is presented on [[Image:new_learning_system3.png|thumb|left|300px|'''Fig. 1: '''educatETH ''E.coli'' System]] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=====.:: Constitutive subsystem ::.=====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=====.:: Learning subsystem ::.=====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=====.:: Reporting subsystem ::.=====<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
====.:: System phases ::.====<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), but not to AHL. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical (GFP) or not (RFP).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! <br />
! aTc<br />
! IPTG<br />
! AHL<br />
! p22cII<br />
! cI<br />
! Reporting <br />
|- <br />
| '''Start'''<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| '''Learning'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| '''Seeing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Memorizing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Testing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| gfp<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| no<br />
| ?<br />
| ?<br />
| rfp<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| gfp<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| no<br />
| ?<br />
| ?<br />
| rfp<br />
|- <br />
<br />
|}<br />
<br />
=====.:: Further thoughts on the system phases ::.=====<br />
<br />
TODO: put Stefan's new text here. Text should be smaller.<br />
<br />
<p>[[Image:LearningSystemOverview.jpg|thumb|right|200px| '''Fig. 2: '''Abstraction of our learning system]]<br />
<br />
A straightforward approach on how to describe learning behavior and adaptive evolution, can be see in Figure 1. We can separate the process in two phases: a <i>training</i> or <i>learning phase</i> (shown in blue), and an <i>application</i> or <i>"real world" phase</i> (shown in pink), and describe our system as a multiple state automaton. The examined system can alter its state according to a certain input/stimuli that it was exposed to in the first phase. In this binary example, the system changes its state from <i>a</i> to <i>b</i> when it is exposed to a first training-phase-input (<i>IT1</i>). Similarly, the system changes its state from <i>a</i> to <i>c</i> when the other training-phase-input (<i>IT2</i>) is applied.<br />
<br />
The principle described above is also valid in the <i>application phase</i>. In the application phase, we have two possibilities for initial system state. Thus, the automaton expands with four more states. Depending on which state the system is at the start of the <i>application phase</i>, and which chemical it is exposed to, it reaches a different final state. State <i>d</i> is reached if the first out of two possible application-phase-inputs (<i>IA1</i>) is applied while the system is at state <i>b</i>. In the end, we can differentiate between the possibilities of the system being trained with one chemical, and being exposed to a different chemical in the application phase. Since we have two different <i>training</i> chemicals, and two different <i>application</i> chemicals, we reach the final number of four states.<br />
<br />
In the following, we elaborate on the two phases of our system:<br />
<ul><br />
<li>How can we describe learning ability with this approach?<br>Define the training-phase-inputs themselves as the information entities to be learned. This implies that after the training phase, the information is permanently stored in the system - ''a memory has been created''. According to its memory, the system will behave differently when it is exposed to a certain stimuli in a later stage.<br />
<br />
<li>If one have a look at ''Figure 1'' once more, one can easily spot similarity to family trees or [http://en.wikipedia.org/wiki/Phylogenetic_tree phylogenetic trees]. This raises the concept of divergent evolution (adaptation). There are several differences to the learning model: First of all, the states don't describe a single living entity with changing characteristics but different populations or species. Secondly, the training-phase-inputs and application-phase-inputs are not related to information inputs but rather to events acting on those populations/species. Thirdly, there is no specific training- and application phase but just two phases with different events/stimuli acting on the populations/species.<br>The following example might show this concept more clearly: Let's say the population of precursor (= ancestor) species ''a'' is splitted into two subpopulations (''a1'' and ''a2'') due to emigration of subpopulation ''a1'' to another geographic region. Application-phase-input 1 (''AI1'') would then equal "emigration" (''a'' -> ''a2'') and application-phase-input 2 (''AI2'') would be "no emigration" (''a'' -> ''a1''). The isolated populations then undergo changes as they (1.) become subjected to dissimilar selective pressures or (2.) they independently undergo genetic drift. When the populations come back into contact, they have evolved such that they are reproductively isolated and are no longer capable of exchanging genes [1]. Therefore, subpopulation ''a1'' has evolved into species ''b'', whereas subpopulation ''a2'' has evolved into species ''c''.<br>This example explains the principle of [http://en.wikipedia.org/wiki/Allopatric_speciation allopatric speciation]. However, our model is not limited to this: Peripatric speciation, parapatric speciation, sympatric speciation or artificial speciation can also be expressed through this model system. Another highly exciting aspect of our model system is, that it can describe '''Evolution without changing the DNA content over time!''' How this can be achieved is explained in more detail in the following sections.<br />
<li>(The automaton of Fig. 1 presents similarities to family trees, or [http://en.wikipedia.org/wiki/Phylogenetic_tree phylogenetic trees]. According to different external stimuli, the initial population divides, and evolves into different species. The proposed automaton model can be used to explain the concepts of peripatric speciation, parapatric speciation, sympatric speciation or artificial speciation e.t.c. However, the most imporant fact, and what has stimulated our research in the area, is, to <i>create a biological system that can evolve without changing its DNA content over time</i>).<br />
</ul></p><br />
<br />
====.:: System parts ::.====<br />
<br />
educatETH ''E.coli'' consists of 11 parts which may be synthesized independently. Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
The system consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!)<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System parts'''<br />
|-<br />
|-<br />
! 1<br />
| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| cI production<br />
| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]<br />
| learning subsystem<br />
| <partinfo>BBa_I739006 SpecifiedComponents</partinfo><br />
|-<br />
! 7<br />
| P22 cII production<br />
| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]<br />
| learning subsystem<br />
| <partinfo>BBa_I739007 SpecifiedComponents</partinfo><br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| ERFP production <br />
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo><br />
|-<br />
! 11<br />
| GFP production <br />
| <br />
| reporting subsystem<br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
! 1<br />
| cI negative / tetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| luxR/HSL positive / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]<br />
| learning subsystem<br />
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| luxR/HSL positive / cI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]<br />
| learning subsystem<br />
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| tetR negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo><br />
|-<br />
! 6<br />
| cI negative / lacI negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
! 1<br />
| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
====.:: References ::.====<br />
<p>[1] <i>Standard Assembly Process</i>, http://partsregistry.org/Assembly:Standard_assembly</p><br><br />
<br />
====.:: To Do ::.====<br />
<br />
=====.:: New ::.=====<br />
<p><ul><br />
<li> Update and correct parts in parts list. Write better in a table<br />
<li> Update and correct full system scheme<br />
<li> Update graph scheme (made by Stefan) using aTc, IPTG instead of it1,2 and ia1,2<br />
<li> Which reporters are active when? I think CFP and YFP are not active only during training. Change text if needed.<br />
<li> Proposed terminology: seeing, memorizing<br />
<li> What are GFP, RFP controlled by? Is the full system scheme correct there?<br />
<li> What are the “double promoters” mentioned?<br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> How was Sven’s standard notation on how to write differently proteins, dna, rna?<br />
<li> Fill in table completely, make it more reading-friendly<br />
<li> Make "In the lab page", replace links.<br />
<li> Put image with 11 system parts (updated one, created by katerina)<br />
</ul></p><br><br />
=====.:: Old::.=====<br />
<p><ul><br />
<li>''Katerina'': 1. Number system parts on both figures for easier reference.<br />
<li>''Katerina'': 2. Add more info on all system parts and link to the ones existing in the registry. Write info on the ones that didn't exist in the registry (with detailed info such as addition of bp's as Christian and Sven had done).<br />
<li>''Katerina'': 3. Add cloning plan. (Christos: Maybe the details will be at the team note's?)<br>Martin: I wouldn't put it here, I mean the cloning plan is really nothing special, it's like an auxiliary calculation for a polynom division... Not exciting and everyone could do it... Please correct me if I'm wrong. By the way I wouldn't write so much about the methods with which we cloned the parts in. These are generally known and every biologist knows them. So I think a jury member who knows all the stuff (which he really should) will be bored if he has to work through the whole assembly and plasmid stuff. I would just say, that we use three Plasmids (with names) for insertion of our system into the bacteria and I would also write which part is on which plasmid, but not more. I would more concentrate on the System, how it's working, what parts we've used and so on....<br />
<li>''Christos'': 1. The picture with the abstraction - maybe we can put better names on the arrows.<br />
<li>''Christos'': 2. We can make figure 1 more specific. Instead of having IT and IA, we can have Chemical 1, Chemical 2, and then again. I think it will be more obvious like that.<br />
<li>''Christos'': 3. Nice descriptions Stefan. However, I made the text less, and kept the original idea, since I felt like it was moving away from the purpose, which is a simple introduction and clarification of concepts. I also removed the pictures. I have backups of everything, so, we can put it back if the others disagree.<br />
<li>''Christos'': 4. Figure 3 needs to be replaced with the new parts.<br />
<li>''Christos'': 5. Are we sure the plasmids that we say are the correct ones? Sven said they were changed, but I didn't really get it.<br>Martin: Yes, they are the right ones... If not, the shit is hitting the fan - albeit in my opinion it has already hit the fan, but not due to the plasmids more due to Genefart...I can really assure you, that they are right. ;-)<br />
</ul></p><br></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZETHZ2007-10-18T13:04:49Z<p>Kdikaiou: fixed link to lab page.</p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<br />
__NOTOC__<br />
<br />
<center><font size = '+2'><b> .:: ETH Zurich - educatETH ''E.coli'' ::. </b></font></center><br><br />
<br />
==='''.:: Introduction ::.'''===<br />
<p> It is the '''3<sup>rd</sup>''' time the [http://www.ethz.ch ETH Zurich] takes part in [https://2007.igem.org/wiki/index.cgi International Genetically Engineered Machine Competition] (iGEM). This year, our combined team of biologists and engineers has undertaken the task of educating <i>E.coli</i> ! [[Image:Educateth_Ecoli.png|thumb|'''Fig. 1:''' The ETH Zurich team undertook the task of engineering a learning mechanism for <i>E.coli</i>.|500px]]More specifically, in our project (Fig. 1), we are trying to create <i>E.coli</i> which have the ability to distinguish between two chemicals they may be exposed to after they have undergone a training phase.</p><br />
<br />
<p>Learning plays a major role for living organisms, as it enables them to survive by adapting to an ever-changing environment. Engineering a simple biological system which exhibits learning behavior is of great interest, as it can support our understanding of this procedure by comparison with natural systems. On the other hand, learning and memorizing plays an equally important role in engineering; from handwriting recognition on PDAs to plain logical circuits storing their binary state on computers, it can be found on numerous everyday life applications. Constructing a biological analogue of a simple memory as known from logic design can hopefully function as a biological building block from which more complex systems may be constructed. We therefore think that educatETH <i>E.coli</i> operates on an exciting interface between engineering and biology.</p><br />
<br />
<p>Stay in this page for an overview of how educatETH <i>E.coli</i> works, the motivation behind it and its possible future applications. If you want to see the biological design of our system and the parts that it consists of, or if you are interested in building it yourself and want to read the lab protocols, the [[ETHZ/Biology | Biology Perspective]] will be of interest to you. If you want to know more on how educatETH <i>E.coli</i> has been modeled and simulated, or on its equivalences to systems such as flip-flops and finite state machines, please visit the [[ETHZ/Engineering | Engineering Perspective]]. You may also want to visit [[ETHZ/Meet_the_team | Meet the Team]] for information regarding the team and [[ETHZ/Pictures | Pictures!]] for our photo gallery. Finally, in [[ETHZ/Internal | Team Notes]] you can read the notes exchanged by the team during preparation for the competition.</p><br />
<br />
====='''.:: Team Members ::.'''=====<br />
KICK PEOPLE TO HAVE A PROPER LINK<br />
<p>As [https://2007.igem.org/wiki/index.cgi iGEM] is a synthetic biology competition, the ETH Zurich team consists of balanced numbers of biology and engineering students. Our team members are: </p><ul> <br />
<li><i>Project advisors</i>: [https://2007.igem.org/User:sven Sven Panke], Joerg Stelling<br />
<li><i>Undergraduate students</i>: [https://2007.igem.org/User:brutsche Martin Brutsche], [https://2007.igem.org/User:kdikaiou Katerina Dikaiou], [https://2007.igem.org/User:Raphael Raphael Guebeli], [https://2007.igem.org/User:hoehnels Sylke Hoehnel], [https://2007.igem.org/Nan_Li Nan Li], [https://2007.igem.org/User:Stefan Stefan Luzi]<br />
<li><i>Graduate students</i>: [http://christos.bergeles.net Christos Bergeles], [http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm], [http://fm-eth.ethz.ch/eth/peoplefinder/FMPro?-db=phonebook.fp5&-format=pf%5fdetail%5fde.html&-lay=html&-op=cn&Typ=Staff&Suche%5fText=kemmer&Suche%5fText%5fpre=kemmer&-recid=3772770936&-find=/ Christian Kemmer], [https://2007.igem.org/User:JoeKnight Joseph Knight], [https://2007.igem.org/User:uhrm Markus Uhr], [http://www.ricomoeckel.de Rico Möckel]<br />
</ul><br />
<p>For more information on the team members, follow the links or visit [[ETHZ/Meet_the_team | Meet the Team]].</p><br />
<br />
<br />
====='''.:: <i>E.coli</i> Intelligence (E.I) - Specifications and analogies ::.'''=====<br />
<br />
<p>How can we make <i>E.coli</i> bacteria able to report us about their environment? Can we teach bacteria to tell if they have seen before a specific chemical? [[Image:System_blended4.png|thumb|350px| '''Fig. 2: '''educatETH ''E.coli'' System ]]</p><br />
<br />
<p>This problem is essentially broken down to constructing a toggle switch which can maintain the state it acquired during a training phase. In Logic Design, this is done using a JK flip-flop with a latch. With this approach, reporting in the testing phase may be implemented with AND gates using the state of the toggle and the current chemical as inputs.<br><br />
(Want to read more about this? Visit the [[ETHZ/Engineering| Engineering Perspective]]!)</p><br />
<br />
<p>Back to biology, a toggle switch has been successfully implemented in [1]. This toggle switch, however, changes states according to one input only as it has one operator site and therefore loses its previous state whenever the system is exposed to a different chemical. This means that it cannot memorize information. To overcome this, we modified the toggle switch in [1] using two operator sites. In this way, a second chemical acting as a “helper” substance present only in the training phase may be used so that the toggle maintains its state during testing.<br> <br />
(Want to see the biological design of our modified toggle? Visit the [[ETHZ/Biology | Biology Perspective]]!)</p><br />
<br />
====='''.:: A short system description ::.'''=====<br />
<br />
<p>educatETH <i>E.coli</i> is able to recognize between two chemical substances (aTC and IPTG) it has previously been exposed with help of an external chemical signal (AHL).<br />
In the first part of the training phase (“meeting”), the system is exposed to one of the two chemicals (aTC and IPTG) and AHL is added, causing a steady system behavior. In the second part of the training phase (“memorizing”), all chemicals but AHL are removed, allowing the system to maintain its state. Finally, in the testing phase (“recognizing”), the system is exposed to any of the two chemicals again. Via comparison of the toggle's steady state with the system response the new chemical causes, the system recognizes if it has been exposed to this chemical before or not. As seen on Table , four system states are possible in the testing phase. We chose to use two reporters to control which chemical educatETH ''E.coli'' is exposed to during the training phase (CFP for , YFP for ) and two others to control whether the system sees the same chemical as it did in the training phase (GFP) or not (RFP). Thus, the system response is determined uniquely. </p><br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! Training <br>chemical <br />
! Testing <br>chemical <br />
! Fluorescence <br>during training<br />
! Fluorescence <br> during testing <br />
|- <br />
| aTc<br />
| aTc<br />
| YFP<br />
| YFP<br />
|- <br />
| aTc <br />
| IPTG<br />
| YFP<br />
| GFP<br />
|- <br />
| IPTG <br />
| aTc <br />
| CFP<br />
| RFP<br />
|- <br />
| IPTG<br />
| IPTG <br />
| CFP<br />
| CFP<br />
|}<br />
<br />
====='''.:: References ::.'''=====<br />
[1]: Toggle Switch<br />
<br />
====='''.:: Acknowledgements ::.'''=====<br />
<ul><br />
<li><p>[http://europa.eu The European Union]</p><br />
<li><p>[http://www.ethz.ch The ETH Zurich]</p><br />
<li><p>[http://www.geneart.com GENEART]</p><br />
</ul><br><br />
====='''.:: Links ::.'''=====<br />
<ul><br />
<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]</p><br />
<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]</p></ul><br><br />
<br />
=='''.:: To Do ::.'''==<br />
====='''.:: New ones ::.'''=====<br />
<br />
<p><ul><br />
<li> How about changing "educatETH <i>E.coli</i> to all italics <i>educatETH E.coli</i><br />
<li> Change the name of p22cII to only cII (its a cI analog) p22 just means that it derives from the phage p22.<br />
<li> IMPORTANT: THE COLOURS ARE TOTALLY WRONG IN THE SIMULATION PART. Please refer to the table on the main page. Changed it just now, it was wrong before!!!<br />
<li> Change banner on top of page. Stefan: Can we please change the banner back to the old version?!? It was MUCH nicer.<br />
<li> Decide on headings type. I like larger headings more, I also like the horizontal line because it separates, put it only on Introduction for you to see how it is.<br />
<li> Possibly put table of contents. <br />
<li> Possibly take educatETH <i>E.coli</i> from Figure.<br />
<li> Change Figure 2.<br />
<li> Possibly change figure caption format from xxx (Fig. x) to Fig.x: xxx. Put second format on Figure 2 for all to check.<br />
<li> Fix table showing possible system states and reporters. Maybe with some color?<br />
<li> Put cross-reference on table.<br />
<li> Align table left<br />
<li> Add which reporters do what in paragraph.<br />
<li> Put (compressed!) team photo instead of Fig.1<br />
<li> Fix paragraph with system description, looks somehow bulky. Maybe italics or bold on important things?<br />
<li> Fix reference on toggle switch.<br />
<li> Possibly remove Introduction heading altogether, and just start with the text.<br />
</ul></p><br />
<br />
====='''.:: Old ones ::.'''=====<br />
<p><ul><br />
<li>Need picture: Bacteria red, Bacteria Green, two pictures showing different exposure to chemicals (''Sylke: see my presentation Sven will hold for me tonight (20.09) -> the FACS guy wants probes of our XFP expressing E.coli - the one who brings it to him can just put it under the microscope and take a few pictures for the wiki'')<br />
<li>Need picture: Einstein ecoli (''Sylke: does Stefan have the layer file?'')<br />
<li>Stupidity: All E. Colis are equal, but our E. Colis are more equal than the others :D<br />
<li>''Katerina'': 2. If we have a separate "Meet the team" page (which is good that we have), we have to make sure that all data about each one of us (short bio+photo) appear also on each one's user page-the same version would be the best, in my opinion.<br />
<li>''Katerina'': 3. Figure 2, bottom right part needs to be a bit larger/more clear, in my opinion, as it's important. (Christos: If you click, then it becomes larger. Will have the same at the bio part as well... Should I make it bigger anyway? I will change it, it is wrong anyway :D)<br />
</ul></p><br />
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</body></html></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZETHZ2007-10-18T13:03:46Z<p>Kdikaiou: Put motivation up.</p>
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<div><center>[[Image:Eth_zh_logo_4.png|830px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
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<br />
__NOTOC__<br />
<br />
<center><font size = '+2'><b> .:: ETH Zurich - educatETH ''E.coli'' ::. </b></font></center><br><br />
<br />
==='''.:: Introduction ::.'''===<br />
<p> It is the '''3<sup>rd</sup>''' time the [http://www.ethz.ch ETH Zurich] takes part in [https://2007.igem.org/wiki/index.cgi International Genetically Engineered Machine Competition] (iGEM). This year, our combined team of biologists and engineers has undertaken the task of educating <i>E.coli</i> ! [[Image:Educateth_Ecoli.png|thumb|'''Fig. 1:''' The ETH Zurich team undertook the task of engineering a learning mechanism for <i>E.coli</i>.|500px]]More specifically, in our project (Fig. 1), we are trying to create <i>E.coli</i> which have the ability to distinguish between two chemicals they may be exposed to after they have undergone a training phase.</p><br />
<br />
<p>Learning plays a major role for living organisms, as it enables them to survive by adapting to an ever-changing environment. Engineering a simple biological system which exhibits learning behavior is of great interest, as it can support our understanding of this procedure by comparison with natural systems. On the other hand, learning and memorizing plays an equally important role in engineering; from handwriting recognition on PDAs to plain logical circuits storing their binary state on computers, it can be found on numerous everyday life applications. Constructing a biological analogue of a simple memory as known from logic design can hopefully function as a biological building block from which more complex systems may be constructed. We therefore think that educatETH <i>E.coli</i> operates on an exciting interface between engineering and biology.</p><br />
<br />
<p>Stay in this page for an overview of how educatETH <i>E.coli</i> works, the motivation behind it and its possible future applications. If you want to see the biological design of our system and the parts that it consists of, or if you are interested in building it yourself and want to read the lab protocols, the [[ETHZ/Biology | Biology Perspective]] will be of interest to you. If you want to know more on how educatETH <i>E.coli</i> has been modeled and simulated, or on its equivalences to systems such as flip-flops and finite state machines, please visit the [[ETHZ/Engineering | Engineering Perspective]]. You may also want to visit [[ETHZ/Meet_the_team | Meet the Team]] for information regarding the team and [[ETHZ/Pictures | Pictures!]] for our photo gallery. Finally, in [[ETHZ/Internal | Team Notes]] you can read the notes exchanged by the team during preparation for the competition.</p><br />
<br />
====='''.:: Team Members ::.'''=====<br />
KICK PEOPLE TO HAVE A PROPER LINK<br />
<p>As [https://2007.igem.org/wiki/index.cgi iGEM] is a synthetic biology competition, the ETH Zurich team consists of balanced numbers of biology and engineering students. Our team members are: </p><ul> <br />
<li><i>Project advisors</i>: [https://2007.igem.org/User:sven Sven Panke], Joerg Stelling<br />
<li><i>Undergraduate students</i>: [https://2007.igem.org/User:brutsche Martin Brutsche], [https://2007.igem.org/User:kdikaiou Katerina Dikaiou], [https://2007.igem.org/User:Raphael Raphael Guebeli], [https://2007.igem.org/User:hoehnels Sylke Hoehnel], [https://2007.igem.org/Nan_Li Nan Li], [https://2007.igem.org/User:Stefan Stefan Luzi]<br />
<li><i>Graduate students</i>: [http://christos.bergeles.net Christos Bergeles], [http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm], [http://fm-eth.ethz.ch/eth/peoplefinder/FMPro?-db=phonebook.fp5&-format=pf%5fdetail%5fde.html&-lay=html&-op=cn&Typ=Staff&Suche%5fText=kemmer&Suche%5fText%5fpre=kemmer&-recid=3772770936&-find=/ Christian Kemmer], [https://2007.igem.org/User:JoeKnight Joseph Knight], [https://2007.igem.org/User:uhrm Markus Uhr], [http://www.ricomoeckel.de Rico Möckel]<br />
</ul><br />
<p>For more information on the team members, follow the links or visit [[ETHZ/Meet_the_team | Meet the Team]].</p><br />
<br />
<br />
====='''.:: <i>E.coli</i> Intelligence (E.I) - Specifications and analogies ::.'''=====<br />
<br />
<p>How can we make <i>E.coli</i> bacteria able to report us about their environment? Can we teach bacteria to tell if they have seen before a specific chemical? [[Image:System_blended4.png|thumb|350px| '''Fig. 2: '''educatETH ''E.coli'' System ]]</p><br />
<br />
<p>This problem is essentially broken down to constructing a toggle switch which can maintain the state it acquired during a training phase. In Logic Design, this is done using a JK flip-flop with a latch. With this approach, reporting in the testing phase may be implemented with AND gates using the state of the toggle and the current chemical as inputs.<br><br />
(Want to read more about this? Visit the [[ETHZ/Engineering| Engineering Perspective]]!)</p><br />
<br />
<p>Back to biology, a toggle switch has been successfully implemented in [1]. This toggle switch, however, changes states according to one input only as it has one operator site and therefore loses its previous state whenever the system is exposed to a different chemical. This means that it cannot memorize information. To overcome this, we modified the toggle switch in [1] using two operator sites. In this way, a second chemical acting as a “helper” substance present only in the training phase may be used so that the toggle maintains its state during testing.<br> <br />
(Want to see the biological design of our modified toggle? Visit the [[ETHZ/Biology | Biology Perspective]]!)</p><br />
<br />
====='''.:: A short system description ::.'''=====<br />
<br />
<p>educatETH <i>E.coli</i> is able to recognize between two chemical substances (aTC and IPTG) it has previously been exposed with help of an external chemical signal (AHL).<br />
In the first part of the training phase (“meeting”), the system is exposed to one of the two chemicals (aTC and IPTG) and AHL is added, causing a steady system behavior. In the second part of the training phase (“memorizing”), all chemicals but AHL are removed, allowing the system to maintain its state. Finally, in the testing phase (“recognizing”), the system is exposed to any of the two chemicals again. Via comparison of the toggle's steady state with the system response the new chemical causes, the system recognizes if it has been exposed to this chemical before or not. As seen on Table , four system states are possible in the testing phase. We chose to use two reporters to control which chemical educatETH ''E.coli'' is exposed to during the training phase (CFP for , YFP for ) and two others to control whether the system sees the same chemical as it did in the training phase (GFP) or not (RFP). Thus, the system response is determined uniquely. </p><br />
<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! Training <br>chemical <br />
! Testing <br>chemical <br />
! Fluorescence <br>during training<br />
! Fluorescence <br> during testing <br />
|- <br />
| aTc<br />
| aTc<br />
| YFP<br />
| YFP<br />
|- <br />
| aTc <br />
| IPTG<br />
| YFP<br />
| GFP<br />
|- <br />
| IPTG <br />
| aTc <br />
| CFP<br />
| RFP<br />
|- <br />
| IPTG<br />
| IPTG <br />
| CFP<br />
| CFP<br />
|}<br />
<br />
====='''.:: References ::.'''=====<br />
[1]: Toggle Switch<br />
<br />
====='''.:: Acknowledgements ::.'''=====<br />
<ul><br />
<li><p>[http://europa.eu The European Union]</p><br />
<li><p>[http://www.ethz.ch The ETH Zurich]</p><br />
<li><p>[http://www.geneart.com GENEART]</p><br />
</ul><br><br />
====='''.:: Links ::.'''=====<br />
<ul><br />
<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]</p><br />
<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]</p></ul><br><br />
<br />
=='''.:: To Do ::.'''==<br />
====='''.:: New ones ::.'''=====<br />
<br />
<p><ul><br />
<li> How about changing "educatETH <i>E.coli</i> to all italics <i>educatETH E.coli</i><br />
<li> Change the name of p22cII to only cII (its a cI analog) p22 just means that it derives from the phage p22.<br />
<li> IMPORTANT: THE COLOURS ARE TOTALLY WRONG IN THE SIMULATION PART. Please refer to the table on the main page. Changed it just now, it was wrong before!!!<br />
<li> Change banner on top of page. Stefan: Can we please change the banner back to the old version?!? It was MUCH nicer.<br />
<li> Decide on headings type. I like larger headings more, I also like the horizontal line because it separates, put it only on Introduction for you to see how it is.<br />
<li> Possibly put table of contents. <br />
<li> Possibly take educatETH <i>E.coli</i> from Figure.<br />
<li> Change Figure 2.<br />
<li> Possibly change figure caption format from xxx (Fig. x) to Fig.x: xxx. Put second format on Figure 2 for all to check.<br />
<li> Fix table showing possible system states and reporters. Maybe with some color?<br />
<li> Put cross-reference on table.<br />
<li> Align table left<br />
<li> Add which reporters do what in paragraph.<br />
<li> Put (compressed!) team photo instead of Fig.1<br />
<li> Fix paragraph with system description, looks somehow bulky. Maybe italics or bold on important things?<br />
<li> Fix reference on toggle switch.<br />
<li> Possibly remove Introduction heading altogether, and just start with the text.<br />
</ul></p><br />
<br />
====='''.:: Old ones ::.'''=====<br />
<p><ul><br />
<li>Need picture: Bacteria red, Bacteria Green, two pictures showing different exposure to chemicals (''Sylke: see my presentation Sven will hold for me tonight (20.09) -> the FACS guy wants probes of our XFP expressing E.coli - the one who brings it to him can just put it under the microscope and take a few pictures for the wiki'')<br />
<li>Need picture: Einstein ecoli (''Sylke: does Stefan have the layer file?'')<br />
<li>Stupidity: All E. Colis are equal, but our E. Colis are more equal than the others :D<br />
<li>''Katerina'': 2. If we have a separate "Meet the team" page (which is good that we have), we have to make sure that all data about each one of us (short bio+photo) appear also on each one's user page-the same version would be the best, in my opinion.<br />
<li>''Katerina'': 3. Figure 2, bottom right part needs to be a bit larger/more clear, in my opinion, as it's important. (Christos: If you click, then it becomes larger. Will have the same at the bio part as well... Should I make it bigger anyway? I will change it, it is wrong anyway :D)<br />
</ul></p><br />
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</body></html></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Meet_the_teamETHZ/Meet the team2007-10-15T14:56:58Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|900px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br />
<br />
__NOTOC__<br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - Meet the team::. </b></font></center><br><br />
<br />
In this page, you can find more information on this year's team of ETH Zurich. If you also want to see photos of ourselves and of the preparation for this year's iGEM, don't forget to see [[ETHZ/Pictures | Pictures!]] as well.<br />
<br />
====='''.:: To Do ::.'''=====<br />
<p><ul><br />
<li> Every one: put a small profile on your page.<br />
<li> Every one: send Christos a photo.<br />
<li> Decide what info will be put here, in which order etc.<br />
</ul></p></div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/EngineeringETHZ/Engineering2007-10-15T14:56:26Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|900px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
__NOTOC__<br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - Engineering Perspective ::. </b></font></center><br><br />
<center><font size = '-1'>[[ETHZ/Model | Model]]&nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Parameters | Parameters]]</font></center><br />
<br />
=====.:: Introduction ::.=====<br />
<br />
<p><br />
Having decided to work on an engineered biological system which exhibits learning, we elaborated on its design. Discussing with the biologists of the team, we realized that what we knew from the field of logic design as JK flip-flop with a latch may be implemented with biological parts using a modified toggle switch. Initial simulations showed us that it was possible to reach the desired behaviour. Therefore, a complete framework of differential equations describing the system was constructed and parameters were searched in the literature. Simulations performed with our new detailed model are very encouraging.<br />
In this page, the equations that model our system are and explained. The values that were chosen for the system parameters are presented and the results of our simulations are analyzed. References are provided at the end of the page. For an introduction to system modeling in synthetic biology, please read our modeling tutorial [[ETHZ/Modeling_Basics|here]].</p><br><br />
<br />
=====.:: System Model ::.=====<br />
<br />
<p>Following the guidelines presented in our modeling guide, we divided the biological system into subsystems, each of which was modelled with a system of differential equations. According to what presented in the [[ETHZ/Biology|Biology Perspective]], our system is composed the following three subsystems:<br><br />
# A subsystem of constitutively produced proteins (see Fig. 1), <br />
# The learning subsystem(see Fig. 2), and <br />
# The reporting subsystem (see Fig. 3). <br />
The first two subsystems interact, and thus, they should be considered together. The third subsystem has no feedback with the other two, as it is only used for producing the appropriate fluorescent proteins.<br />
<br />
The subsystem of the constitutively produced proteins serves as a regulatory system, and can be modeled with the following three decoupled partial differential equations (see Fig. 1):<br />
<br />
[[Image:constitutive_part.png|center|thumb|Subsystem 1: Constitutively produced proteins (Fig. 1)|340px]]<br />
<br />
The second subsystem is the main part of our model. It stores the information concerning the learned information for the chemical of interest, and drives the production of the appropriate reporter during the recognition phase. It is actually a toggle switch that reaches its steady state depending on the chemical that it has been previously exposed to (see Fig. 2):<br />
<br />
[[Image:toggle_part_b.png|center|thumb|Subsystem 2: Basic learning subsystem (toggle) (Fig. 2)|900px]]<br />
<br />
The third subsystem reports the state that our system is, during the different phases of learning and recognition. During the learning phase, this subsystem reports which chemical the cells are exposed to, and during the recognition phase, it reports if the cells recognize the chemicals that they are currently exposed to (see Fig. 3):<br />
<br />
[[Image:reporting_part_b.png|center|thumb|Subsystem 3: Reporting subsystem (Fig. 3)|900px]] <br />
<br />
Note that the three constitutively produced proteins LacI, TetR and LuxR exist in two different forms; as free proteins and in complexes they build with IPTG, aTc and AHL, respectively. We need to model this complex-forming procedure, with another set of differential equations (Fig. 4):<br />
<br />
[[Image:allosteric_part.png|center|thumb|Allosteric regulation (see Fig. 4)|240px]]<br />
<br />
In order to have meaningful results from our simulations, we browsed through the literature in order to find appropriate values for our parameters. We reduced our parameter space by joining parameters together, and we gave reasonable estimates, for the values that could not be extracted from available publications. Since this is a hard part that every team has to face, we present the table with the chosen parameters below:<br />
<br />
[[Image:parameters.png|center|thumb|Parameters of the model (Fig. 5)|800px]]<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Parameter <br />
! Value<br />
! Description<br />
! References<br />
|-<br />
| c<sub>1</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of<br> constitutive promoter <br>(per gene)<br />
| Estimate<br />
|-<br />
| c<sub>2</sub><sup>max</sup><br />
| 0.01 [mM/h]<br />
| max. transcription rate of<br> luxR-activated promoter <br>(per gene)<br />
| Estimate<br />
|-<br />
| l<sup>hi</sup><br />
| 25<br />
| high-copy plasmid number<br />
| Estimate<br />
|-<br />
| l<sup>lo</sup><br />
| 5<br />
| low-copy plasmid number<br />
| Estimate<br />
|-<br />
| a<sub>p22cII,LacI</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> p22cII/LacI-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>p22cII</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> p22cII-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>cI,TetR</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> cI/TetR-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>cI</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> cI-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>p22cII,TetR</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> p22cII/TetR-inhibited genes<br />
| Discussion<br />
|-<br />
| a<sub>cI,LacI</sub><br />
| 0.1 - 0.2<br />
| basic production of<br> cI/TetR-inhibited genes<br />
| Discussion<br />
|-<br />
| d<sub>LacI</sub><br />
| 2.31e-3 [pro sec]<br />
| degradation of lacI<br />
| [10]<br />
|-<br />
| d<sub>TetR</sub><br />
| 1e-5 [pro sec]/2.31e-3 [pro sec]<br />
| degradation of TetR<br />
| [9], [10]<br />
|-<br />
| d<sub>LuxR</sub><br />
| 1e-3 - 1e-4 [per sec]<br />
| degradation of LuxR<br />
| [6]<br />
|-<br />
| d<sub>cI</sub><br />
| 7e-4 [per sec]<br />
| degradation of cI<br />
| [7]<br />
|-<br />
| d<sub>p22cII</sub><br />
| <br />
| degradation of p22cII<br />
|-<br />
| d<sub>YFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of YFP<br />
| suppl. mat. to Ref. [8]<br />
|-<br />
| d<sub>GFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of GFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>RFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of RFP<br />
| in analogy to YFP<br />
|-<br />
| d<sub>CFP</sub><br />
| 6.3e-3 [per min]<br />
| degradation of CFP<br />
| in analogy to YFP<br />
|-<br />
| K<sub>LacI</sub><br />
| 1.3e-3 - 2e-3 [mM/h]<br />
| lacI repressor <br>dissociation constant<br />
| [2], [5]<br />
|-<br />
| K<sub>IPTG</sub><br />
| 1.5e-10 [mM/h]<br />
| IPTG-lacI repressor <br>dissociation constant<br />
| [5]<br />
|-<br />
| K<sub>TetR</sub><br />
| 5.6 (+-2) [nM-1]<br />
| tetR repressor <br>dissociation constant<br />
| [1]<br />
|-<br />
| K<sub>aTc</sub><br />
| 1120 (+-400) [nM-1]<br />
| aTc-tetR repressor <br>dissociation constant<br />
| [1], [3]<br />
|-<br />
| K<sub>LuxR</sub><br />
| <br />
* 0.003 [mM/s]<br />
* 55 - 520 [nM]<br />
| luxR activator <br>dissociation constant<br />
| [6]<br />
|-<br />
| K<sub>AHL</sub><br />
| <br />
* 0.009 [mM/s] - 0.1 [mM/s]<br />
* 0.09 - 1 [&#181;M]<br />
| AHL-luxR activator <br>dissociation constant<br />
| [6]<br />
|-<br />
| K<sub>cI</sub><br />
| 2e-3 [mM/h]<br />
| cI repressor <br>dissociation constant<br />
| [5]<br />
|-<br />
| K<sub>p22cII</sub><br />
| <br />
| p22cII repressor <br>dissociation constant<br />
|-<br />
| n<sub>LacI</sub><br />
| 1<br />
| lacI repressor <br>Hill cooperativity<br />
| [5]<br />
|-<br />
| n<sub>IPTG</sub><br />
| 2<br />
| IPTG-lacI repressor <br>Hill cooperativity<br />
| [5]<br />
|-<br />
| n<sub>TetR</sub><br />
| 3<br />
| tetR repressor <br>Hill cooperativity<br />
| [3]<br />
|-<br />
| n<sub>aTc</sub><br />
| 2 (1.5-2.5)<br />
| aTc-tetR repressor <br>Hill cooperativity<br />
| [3]<br />
|-<br />
| n<sub>LuxR</sub><br />
| 2<br />
| luxR activator <br>Hill cooperativity<br />
| [6]<br />
|-<br />
| n<sub>AHL</sub><br />
| 1<br />
| AHL-luxR activator <br>Hill cooperativity<br />
| [3]<br />
|-<br />
| n<sub>cI</sub><br />
| 1.9<br />
| cI repressor <br>Hill cooperativity<br />
| [5]<br />
|-<br />
| n<sub>p22cII</sub><br />
| <br />
| p22cII repressor <br>Hill cooperativity<br />
|-<br />
|}<br />
<br />
<br />
<br />
</p><br><br />
<br />
=====<b>.:: Simulations ::.</b><br>=====<br />
<br />
=====.:: References ::.=====<br />
<p><br />
[1] Weber W., Stelling J., Rimann M., Keller B., Daoud-El Baba M., Weber C.C., Aubel D., and Fussenegger M., <i>"A synthetic time-delay circuit in mammalian cells and mice"</i>, Proceedings of the National Academy of Sciences, vol. 104, no. 8, pp. 2643, 2007.<br><br />
[2] Setty Y., Mayo AE, Surette MG, and Alon U., <i>"Detailed map of a cis-regulatory input function"</i>, Proceeding of the National Academy of Sciences, vol. 100, no. 13, pp. 7702--7707, 2003.<br><br />
[3] Braun D., Basu S., and Weiss R., <i>"Parameter Estimation for Two Synthetic Gene Networks: A Case Study"</i>, IEEE Int'l Conf. Acoustics, Speech, and Signal Processing 2005, vol. 5, 2005.<br><br />
[4] Fung E., Wong W.W., Suen J.K., Bulter T., Lee S., and Liao J.C., <i>"A synthetic gene--metabolic oscillator"</i>, Nature, vol. 435, no. 7038, pp. 118--122, 2005, supplementary material.<br><br />
[5] Iadevaia S., and Mantzaris N.V., <i>"Genetic network driven control of PHBV copolymer composition"</i>, Journal of Biotechnology, vol. 122, no. 1, pp. 99--121, 2006.<br><br />
[6] Goryachev AB, Toh DJ, and Lee T., <i>"Systems analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constants"</i>, Biosystems, vol. 83, no. 2-3, pp. 178--187, 2006.<br><br />
[7] Arkin A., Ross J., and McAdams H.H., <i>"Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-Infected Escherichia coli cells"</i>, Genetics, vol. 149, no. 4, pp. 1633--1648, 1998.<br><br />
[8] Colman-Lerner A., Chin T.E., and Brent R., <i>"Yeast Cbk1 and Mob2 Activate Daughter-Specific Genetic Programs to Induce Asymmetric Cell Fates"</i>, Cell, vol. 107, no. 6, pp. 739--750, 2001.<br><br />
[9] Becskei A., and Serrano L., <i>"Engineering stability in gene networks by autoregulation"</i>, Nature, vol. 405, no.6786, pp.590--593, 2000.<br><br />
[10] Tuttle L.M., Salis H., Tomshine J., and Kaznessis Y.N., <i>"Model-Driven Designs of an Oscillating Gene Network"</i>, Biophysical Journal, vol. 89, no. 6, pp. 3873--3883, 2005.<br><br />
<br />
</p><br><br />
[http://www.pnas.org/cgi/content/abstract/104/8/2643 &#91;1&#93; Weber W et al.] <i>"A synthetic time-delay circuit in mammalian cells and mice"</i>, P Natl Acad Sci USA 104(8):2643-2648, 2007<br /><br />
[http://www.pnas.org/cgi/content/full/100/13/7702?ck=nck &#91;2&#93; Setty Y et al.] <i>"Detailed map of a cis-regulatory input function"</i>, P Natl Acad Sci USA 100(13):7702-7707, 2003<br /><br />
[http://ieeexplore.ieee.org/iel5/9711/30654/01416417.pdf &#91;3&#93; Braun D et al.] <i>"Parameter Estimation for Two Synthetic Gene Networks: A Case Study"</i>, ICASSP 5:769-772, 2005<br /><br />
[http://www.nature.com/nature/journal/v435/n7038/suppinfo/nature03508.html &#91;4&#93; Fung E et al.] <i>"A synthetic gene--metabolic oscillator"</i>, Nature 435:118-122, 2005 (supplementary material)<br /><br />
[http://dx.doi.org/10.1016/j.jbiotec.2005.08.030 &#91;5&#93; Iadevaia S and Mantzais NV] <i>"Genetic network driven control of PHBV copolymer composition"</i>, J Biotechnol 122(1):99-121, 2006<br /><br />
[http://dx.doi.org/10.1016/j.biosystems.2005.04.006 &#91;6&#93; Goryachev AB et al.] <i>"Systems analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constants"</i>, Biosystems 83(2-3):178-187, 2004<br /><br />
[http://www.genetics.org/cgi/content/abstract/149/4/1633 &#91;7&#93; Arkin A et al.] <i>"Stochastic kinetic analysis of developmental pathway bifurcation in phage λ-Infected Escherichia coli cells"</i>, Genetics 149: 1633-1648, 1998<br /><br />
[http://download.cell.com/supplementarydata/cell/107/6/739/DC1/index.htm &#91;8&#93; Colman-Lerner A et al.] <i>"Yeast Cbk1 and Mob2 Activate Daughter-Specific Genetic Programs to Induce Asymmetric Cell Fates"</i>, Cell 107(6): 739-750, 2001 (supplementary material)<br /><br />
[http://www.nature.com/nature/journal/v405/n6786/abs/405590a0.html &#91;9&#93; Becskei A and Serrano L] <i>"Engineering stability in gene networks by autoregulation"</i>, Nature 405: 590-593, 2000<br /><br />
[http://www.biophysj.org/cgi/content/full/89/6/3873?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1&FIRSTINDEX=0&volume=89&firstpage=3873&resourcetype=HWCIT &#91;10&#93; Tuttle et al.] <i>"Model-Driven Designs of an Oscillating Gene Network"</i>, Biophys J 89(6):3873-3883, 2005<br /><br />
[http://www.pnas.org/cgi/reprint/99/2/679 &#91;11&#93; McMillen LM et al.] <i>"Synchronizing genetic relaxation oscillators by intercell signaling"</i>, P Natl Acad Sci USA 99(2):679-684, 2002<br /><br />
[http://www.nature.com/nature/journal/v434/n7037/full/nature03461.html &#91;12&#93; Basu S et al.] <i>"A synthetic multicellular system for programmed pattern formation"</i>, Nature 434:1130-1134, 2005<br /><br />
<br />
=====.:: To Do ::.=====<br />
<br />
*''Christos'': 1. Add gifs concerning the simulations<br />
*''Christos'': 2. Remove the table with the parameters, once we have satisfying values.<br />
*''Katerina'': 1. "The third subsystem has no feedback with the other two, as it is only used for producing the appropriate fluorescent proteins." Is this really true? gfp and rfp are situated in bigger parts with functionality. I think this needs to be rewritten more clearly.</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/InternalETHZ/Internal2007-10-15T14:55:56Z<p>Kdikaiou: </p>
<hr />
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<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+2'><b> .:: ETH Zurich - EducatETH ''E. coli'' ::. </b></font></center><br><br />
<br />
=='''.:: Synthetic Biology Boot camp ::.'''==<br />
To make everybody familiar with synthetic biology concepts and to assist coming up with ideas for an interesting project, we will read and present publications on important topics. The list to be covered is the following: <br />
<br />
* Introduction to synthetic biology (Markus, Martin)<br />
* DNA de novo design (Rico, Raphael)<br />
* DNA circuits (Christian, Nan))<br />
* Hysteresis (Tim, Sylke)<br />
* Oscillators (Christos, Joe, Katerina and Markus, Martin)<br />
* Zinc fingers (Sylke)<br />
* Noise in single cell measurements<br />
* Distance communication (Rico, Raphael)<br />
* Parameter manipulations (Christos, Joe, Katerina)<br />
* Orthogonal systems (Christos, Joe, Katerina)<br />
* Minimal genomes (Markus, Martin)<br />
* RNA regulators (Rico, Raphael)<br />
<br />
All presentations have been uploaded in the known web space. You may also contact the people who did it if you need additional information.<br />
<br />
=='''.:: Brainstorming ::.'''==<br />
<br />
==='''.:: Brainstorming sessions::.'''===<br />
During the first two weeks of July, the team has to come up with a project. The team is divided into three groups, which will brainstorm individually. Sven and Joerg are available on the 4th and 5th of July in case groups needs to consult them. The objective is that each group comes up with many fancy ideas. To facilitate this, keep in mind the following "brainstorming rules":<br />
<br />
# Defer judgment - the rules of nature don't apply<br />
# Encourage wild ideas<br />
# Build on the ideas of others<br />
# Be visual<br />
# Go for quantity<br />
# Stay focused on topic<br />
<br />
==='''.:: Preliminary ideas ::.'''===<br />
<br />
* '''PID Controller''': Design a PID controller out of biological elements. The P component can be a simple output to a regulatory protein, and the I component can be the overall protein production at a time period. What can the D component be?<br />
* '''Motion Detector''': Cells are grown on a petri dish. Below the dish, moving images are displayed. A 3-state automaton is proposed. Output A is created when light is present. Output B is created when light is absent. Moving patterns will cause some cells to create both outputs over time. This will result in some “inspector” cells producing output C, by collecting outputs A and B.<br />
* '''Analog-to-Digital Converter''': Compare the level of protein concentration with thresholds, and digitize the output.<br />
* '''Neural Network''': Create a sort of biological neural network with bacteria. We should address the issue of learning, and find a way to incorporate the feedback in the cell decision making process. Directed evolution can be a sort of feedback, but we want to avoid this. (This idea was the basis for the “learning project”)<br />
* '''Paramedic Cells''': Some cells are able to detect signals coming from other cells, and create food for them, or create proteins in order to save them and make them function better.<br />
* '''Cell Batteries''': Cells are able to create and store large quantities of ATP, during a “storing process”. Afterwards, they can detect a signal and give back all the energy they stored, in a short burst, like a capacitor. Other ideas are that the cells can “blow up” and emit large amounts of GFP, based on the ATP that they have accumulated.<br />
* '''Flashing Bacteria''': Cells are grown on a light pattern. The cells that are on the bright parts of the image are oscillating in phase, while the others are remaining dark. This results in the observation of a flashing pattern.<br />
* '''Biocam''': Visible to Fluorescent light converter.<br />
* '''BioCD''': “Print” cells on a film, then read them out and “reconstruct” the original data. Basically, it is an analog to digital converter, followed by a system that can interpret the digitized data. (This idea was the basis for the “Music of life project”, where cells would produce fluorescent proteins based on an analog input. Then, the amount and type of fluorescence would code some music).<br />
* '''Clock''': A follow-the-leader system. We have to groups of cells. The first group creates something that repels the second group. The second group creates a protein that attracts the first group. This way, they first group wants to “catch” the second group, whereas the second group wants to “avoid” the first group. This results in them moving around. We can say that the second group is the leader, and the first group exhibits a "follow-the-leader” behavior.<br />
* '''Sensors''': Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.<br />
<br />
==='''.:: Preferred projects::.'''===<br />
Three ideas of the above are chosen for further examination. The team will be again split up in three groups (different than before, to make sure that new ideas come up with mxing of people). Each group has to come up with an initial system, with remarks on its feasibility and coolness. Our results<br />
will be presented to all team members, so that potential projects may be limited down to two and subsequently to<br />
one. The preferred projects are: <br />
<br />
# '''Music of Life''': The basic idea is that instead of having an analog-to-digital converter with four outputs (three fluorescent proteins, and no output), we can have two switches. When switch A is on, RFP is produced. When switch B is on, GFP is produced. When both switches A and B are on, a yellowish output is observed. By recording these outputs, we can later create music, by assigning each fluorescent protein to a chord. For example, RFP would correspond to a G chord. The strength of the fluorescence can signify the strength of the chord. If the cells are placed on a spinning disk, we can have something like a vinyl player. A camera is observing the cells, and music is created on the fly. <br />
# '''Learning''': Based on the idea of the neural network, we want to create a biological system, where the cells can learn a specific behavior. In order to simplify the system, we decided that the cells can learn to recognize a specific type of other cells. We divide the process in a learning phase, and a recognition phase. First, cells A are put together with cells B. Then, cells A are “learning” to recognize cells B. If afterwards they are put in a petri dish with cells B, they will emit GPF. Otherwise, they will stay dark.<br />
<br />
==='''.:: Final project::.'''===<br />
<br />
The chosen project is a modified version of Learning which was presented at the last meeting. The system proposed is modified as in its current state may be implemented with two switches only, something which is not exciting enough. Keeping the idea of learning, and of training and testing phases, we have come up with EducatETH <i>E.coli</i>.<br />
<br />
=='''.:: Task List ::.'''==<br />
<br />
==='''.:: Project Task List ::.'''===<br />
<br />
The things to do, from the most pressing (timewise), to the least pressing (timewise) is below. Please put your name next to the task that you believe that you can undertake.<br />
<br />
# '''Team descriptions (overdue) and team photograph''' <br> ''Christian'': I guess I am the only semiprofessional photographer of the group. I can do some group pictures etc. but for this we need some ideas... - I would like to do something special. I also made the group-pics of the Synth. Biology 3.0 conference. Some references ;-) : [http://www.fotocommunity.de/pc/pc/mypics/461397] <br> ''Raphael'': What about a short movie of us?<br />
# '''Team rosters due (1.9.)'''<br> ''Martin'': Does somebody know, what exactly should be done here? <br> ''Nan'': A list of team members, including some basic personal info.? (e.g. nationality, background, pet peeves...? )<br>''Katerina'': Guys, to make this easier, either write things about you on your personal page on the wiki or link to a page about you. This way it'll be easy to put it all together afterwards.<br />
# '''Labwork (parts have to be at the registry in Boston on 26.10.)''' <br> ''Joe'': I can be in the lab at least 2 evenings a week and some times through the weekdays. <br> ''Martin'': From Monday I can work every day for the whole day. At the moment I only work for several hours... <br> ''Rico'': I have my exam on Tuesday. Afterwards I can assist.<br> ''Christian'': I can do the introduction of the polylinker into the vectors beside my normal labwork on the Hoenggerberg. I could also do the whole biobrick assembly if you want this (I will go on holiday from the 5.10-24.10.07) <br> ''Raphael'': That's the part where I will mainly contribute, from 14.09. on I can work several days/week<br> ''Christos'': I can assist at the afternoons, if needed. <br> ''Katerina'': 7.09 - 30.09 generally plenty of time, apart from when I do my semester project presentation, will keep you posted when that is. <br> ''Sylke'': I'll not be available until 14th of October but from then on I can work several days per week fulltime.<br />
# '''Testing/ Analysis/ Detection (due to 02.11.)''' <br> ''Sylke'': Meeting with Alfredo Franco-Obregon (FACS guy from center) on 18.09. concerning devices available for detection of GFP/ derivatives.<br />
# '''Simulation and sensitivity analysis'''<br> ''Martin'': From 10. Sept. I've got plenty time to work on it. I think Markus would join here too ;-) <br> ''Tim'': I can contribute in running stuff and help identify parameters from literature <br> ''Rico'': I can do simulations, sensitivity analysis. <br> ''Nan'': I would like to do simulations and sensitivity analysis. <br> ''Christos'': Yeap, I guess I can be here too. I will check some toolboxes to automate things, this weekend. <br> ''Katerina'': Want to help (parameter identification, programming, sensitivity analysis). Discuss in upcoming meeting tasks, versioning and ask Christian about parameters.<br />
# '''Presentation''' <br> ''Joe'': I'm American... I can sale anything.<br> ''Martin'': I'm bad in Layout stuff, but maybe I can help as an idea supplier or so. <br> ''Rico'': I like giving presentations. For preparation we will need a mixture of different background and excellent pictures!!! <br> ''Christian'': I can provide the molecular biology knowledge and part. <br> ''Nan'': I can work on the slides. <br> ''Christos'': I like this part, I guess everyone will contribute anyway...<br> ''Katerina'': I believe I can be of help in structure, layout and fancy stuff. Could help train a bit the people we decide to do the presentation (question answering, style). I also think that Joe and Christos could be good for doing the presentation (structured, pleasant voices and lively), can discuss this in a meeting. <br> ''Sylke'': I can help with the bio-part and I'm good at doing graphics and stuff. Can be a help when it needs to look good :-) <br />
#''' Poster''' <br> ''Joe'': See 5. above <br> ''Rico'': I can help. <br> ''Christian'': I can provide the molecular biology knowledge and part. <br> ''Nan'': Partly art. I will help. <br> ''Christos'': You can sell, but can you trick? lol :)<br> ''Katerina'': Not my strong point, but have an eye for typos, fonts, layout etc, so can help in final checking. <br> ''Sylke'': See above. Good at layout.<br />
# '''Wiki (Project and part documentation due on 26.10)''' <br> ''Martin'': See the points above, from next Monday I will give everything, now I'm doing my best... <br> ''Rico'': I can help. I guess this will have to contain the materials that we will also use for poster and presenttion anyway. <br> ''Nan'': Shouldn't it be updated with every going on process? <br> ''Christos'': I am trying to put stuff in, as it comes along. I will update the bio pages with the presentation material, this weekend (I hope). <br> ''Katerina'': You guys have done a great job so far, will help with whatever needed.<br />
# '''T-Shirts''' <br> ''Sylke'': in print (2007-09-24)</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/Biology/LabETHZ/Biology/Lab2007-10-15T14:55:37Z<p>Kdikaiou: </p>
<hr />
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<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E. coli</i> - In the lab ::. </b></font></center><br><br />
<br />
In this page, you can find information on laboratory conducted to construct EducatETH <i>E.coli</i>. The system parts are presented again, their assembly into plasmids and the cloning plan are explained and all lab notes taken by the ETH Zurich team are accessible. If you are trying to construct EducatETH <i>E.coli</i> at your lab, the section [https://2007.igem.org/ETHZ/Biology/Lab#.::_Problems_we_faced_::. Problems we faced] might be useful to you. If you want to see the whole biological design of the system, please visit the [[ETHZ/Biology | Biology Pespective]]. Finally, photos of our lab experience are accessible under [[ETHZ/Pictures | Pictures!]]<br />
<br />
Todo: decide what happens with lab book ([https://2007.igem.org/ETHZ/Lab_book here])<br />
<br />
==='''.:: List of system building blocks ::.'''===<br />
<br />
Here is a list of all the registry parts we used as bulding blocks for our system parts. This list has to be updated and extended for the new, concatenated parts that the ETH Zurich has submitted to the registry.<br />
<br />
# [http://partsregistry.org/Part:BBa_B0034 B0034]<br />
# [http://partsregistry.org/Part:BBa_R0062 R0062]<br />
# [http://partsregistry.org/Part:BBa_R0053 R0053]<br />
# [http://partsregistry.org/Part:BBa_J23100 J23100]<br />
# [http://partsregistry.org/Part:BBa_J37033 J37033]<br />
# [http://partsregistry.org/Part:BBa_E0434 E0434]<br />
# [http://partsregistry.org/Part:BBa_B0015 B0015]<br />
# [http://partsregistry.org/Part:BBa_Q04400 Q04400]<br />
# [http://partsregistry.org/Part:BBa_R0010 R0010]<br />
# [http://partsregistry.org/Part:BBa_E0422 E0422]<br />
# [http://partsregistry.org/Part:BBa_R0040 R0040]<br />
# [http://partsregistry.org/Part:BBa_R0051 R0051]<br />
# [http://partsregistry.org/Part:BBa_Q04121 Q04121]<br />
# [http://partsregistry.org/Part:BBa_C0053 C0053]<br />
# [http://partsregistry.org/Part:BBa_Q04510 Q4510]<br />
<br />
=='''.:: Cloning plan::.'''==<br />
<br />
==='''.:: Parts assignment into plasmids::.'''===<br />
<br />
Three plasmids are used for the EducatETH <i>E.coli</i> system parts as follows:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Plasmids and contents'''<br />
|-<br />
! plasmid !! resistance !! copy type!! contents !! comments<br />
|-<br />
<br />
| [[ETHZ/pbr322| pbr322]] || ampicillin || high || 1,2,3 || constitutive subsystem<br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || chloramphenicol|| low || 4,5,8,9 || reporting subsystem<br />
|-<br />
<br />
| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| low || 6,7,10,11 || learning subsystem, reporting subsystem<br />
|-<br />
|}<br />
<br />
It is important to insert parts responsible for the production of fluorescent proteins in low copy plasmids, as they are potentially harmful for the cell. Unfortunately, working with low copy plasmids makes the procedure more demanding in the lab.<br />
<br />
==='''.:: Linkers::.'''===<br />
<br />
Because the plasmids used were not standard plasmids found in the registry, but came from the lab where we work, linkers compatible with the standard BioBrick assembly have to be used in order to work with them. The list of all linkers is the following:<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
|+'''Linkers for plasmids'''<br />
|-<br />
! Linker!! Plasmid<br />
|-<br />
<br />
| [[ETHZ/pbr322-1| pbr322-1]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-2| pbr322-2]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-3| pbr322-3]]|| pbr322 <br />
|-<br />
| [[ETHZ/pbr322-4| pbr322-4]]|| pbr322 <br />
|-<br />
<br />
| [[ETHZ/pck01| pck01]] || pck01 <br />
|-<br />
| [[ETHZ/pCK01-2| pck01-2]] || pck01 <br />
|-<br />
<br />
| [[ETHZ/pacyc177-1| pacyc177-1]] || pacyc177<br />
|-<br />
| [[ETHZ/pacyc177-2| pacyc177-2]]|| pacyc177<br />
|-<br />
|}<br />
<br />
Note that four linkers are tested for pbr322, as two are used for the tetracycline-resistance version of pbr322 and two are used for the ampicillin-resistnace version.<br />
<br />
==='''.:: Procedure::.'''===<br />
<br />
The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. [[Image:Assembly _process.png|thumb|300px|DNA assembly process ([1]) '''(Fig. 4)''']] Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted ''before'' the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the ''donor'' and the plasmid accepting the new part will be referred to as the ''acceptor''. Composite pars made of parts '''a''' and '''b''' are denoted '''a.b'''.<br />
<br />
===== '''.::Plasmid 1 ''(pbr322ap)''::.''' =====<br />
<br />
# Put parts 1,2,3 in pbr322ap plasmids. <br />
# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.<br />
# Merge plasmid containing part '''1''' ''(donor)'' with plasmid containing composite part '''2.3''' ''(acceptor)''. You should get a plasmid containing a '''1.2.3''' composite part.<br />
<br />
===== '''.::Plasmid 2 ''(pck01cm)''::.''' =====<br />
<br />
# Put parts 4,5,8,9 in pck01cm plasmids. <br />
# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.<br />
# Merge plasmid containing part '''8''' ''(donor)'' with plasmid containing part '''9''' ''(acceptor)''. You should get a plasmid containing a '''8.9''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''4.5''' ''(donor)'' with plasmid containing composite part '''8.9''' ''(acceptor)''. You should get a plasmid containing a '''4.5.8.9''' composite part.<br />
<br />
===== '''.::Plasmid 3 ''(pacyc177km)''::.''' =====<br />
<br />
# Put parts 6,7,10,11 in pacyc177km plasmids. <br />
# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.<br />
# Merge plasmid containing part '''10''' ''(donor)'' with plasmid containing part '''11''' ''(acceptor)''. You should get a plasmid containing a '''10.11''' composite part. ''Note'': this step can be done simultaneously with the above.<br />
# Merge plasmid containing composite part '''6.7''' ''(donor)'' with plasmid containing composite part '''10.11''' ''(acceptor)''. You should get a plasmid containing a '''6.7.10.11''' composite part.<br />
<br />
=='''.:: Problems we faced ::.'''==<br />
<br />
=='''.:: Lab book ::.'''==<br />
<br />
===='''.:: Week 1 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 06. Aug. 2007 <br />
|<br />
| <br />
* Preparing the Solutions <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Tue, 07. Aug. 2007 <br />
| <br />
|<br />
* Prepare competent cells for all parts<br />
* Transformation of all the parts <br />
| Sylke<br>Raphael<br>Stefan<br>Markus<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Wed, 08. Aug. 2007 <br />
| <br />
| <br />
* Preparing the grown cultures (12) for the MINIPREP <br> (o/n cultures) <br />
| Raphael<br>Stefan<br />
|-<br />
| Thu, 09. Aug. 2007 <br />
| <br />
| <br />
* MINIPREP of the grown (10) o/n cultures<br />
* Gelelectrophoresis of the grown cultures (step: 0.8% Agarose) <br />
| Raphael<br>Stefan<br>Martin<br>Christos<br>Joe<br />
|-<br />
| Fri, 10. Aug. 2007 <br />
| <br />
| <br />
* 7 working parts/plasmids (step after "DIGESTS"): <br> (B0034, R0062, R0053, E0434, B0015, R0010, E0422)<br />
* 4 parts/plasmids minipreped: <br> (R0040, R0051, Q04121, C0053)<br />
|<br />
Christos <br> Markus <br> Stefan<br />
|-<br />
| Sat, 11. Aug. 2007 <br />
| <br />
| no labwork <br />
|<br />
|-<br />
| Sun, 12. Aug. 2007 <br />
|<br />
| labwork cancelled<br />
|<br />
|}<br />
<br />
===='''.:: Week 2 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 13. Aug. 2007 <br> start at 3 pm <br />
|<br />
* Prepare competent cells<br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
* Control Restrictions (step after "MINIPREP") <br>R0040, R0051, Q04121, C0053<br />
| <br />
* o/n culture (E.Coli Top10)<br />
* Control Restrictions (didn't work)<br />
| Martin<br> Markus <br> Christos <br> Tim <br><br />
|-<br />
| Tue, 14. Aug. 2007 <br />
| Morning Shift: <br><br />
* Start Preparing competent cells (for J23100, J37033, Q04400, Q04510) <br> <br />
Evening Shift: <br><br />
* Transformations of J23100, J37033, Q04400, Q04510<br />
| Morning Shift: <br><br />
* Prepared competent cells (stored in the -80°C freezer in the basement) <br><br />
Evening Shift: <br><br />
* Transformation of J23100, J37033, Q04400, Q04510 and R0040, R0051, Q04121, C0053 (in the 37°C incubator until Wednesday) <br />
* Prepared new Liquid LB, LB Agar (both in the autoclave), Agarose Gel with concentrations of 0.8% and 2.4% <br />
|Morning Shift (9am-1pm?): <br> Markus <br> Tim <br> Evening Shift (5pm-...):<br> Martin <br> Christos<br />
|-<br />
| Wed, 15. Aug. 2007 <br />
| <br />
* Ligation (step: "LINK ASSEMBLY"): <br> R0053 + E0422 <br> R0010 + E0422 <br> R0010 + E0434 <br> S/P: R0053, R0010 <br> X/P: E0422, E0434 <br />
|<br />
* Ligation didn't work due to bad quality of enzymes (probably) <br />
| From 12:<br> Martin<br>Markus<br><br><br />
|-<br />
| Thu, 16. Aug. 2007 <br />
| <br />
* Miniprep (J23100, J37033, Q04400, Q04510, R0040, R0051, Q04121, C0053)<br />
* Transformation of #13 and #14<br />
| <br />
* Miniprep of #4 (J23100), #5 (J37033), #8 (Q04400), #11 (R0040), #12 (R0051), #15 (Q04510) <br> One batch is miniprepped (after step 19 in the miniprep procedure) and a second batch is frozen as a backup (which is to be miniprepped from step 3 on)<br />
* Transformation of #13 (Q04121) and #14 (C0053) <br> Numbers #13 and #14 are now growing in the 37°C incubator (step 13 in the transformation procedure)<br />
|<br />
Markus<br>Christos<br>(Martin)<br />
|-<br />
| Fri, 17. Aug. 2007 <br />
|<br />
* o/n of #13 and #14<br />
* Check whether miniprep of parts #4 #5 #8 #11 #12 (#13 #14) #15 was successful<br />
| <br />
* #13 and #14 didn't grow<br />
* # 4, 8 and 11 had the plasmid, they were streaked out new on plates, that we have them now on plates<br />
* New white pipette tips prepared (autoclave)<br />
* New bottles of Liquid LB and LB Agar prepared (autoclave)<br />
| Martin<br />
|-<br />
| Sat, 18. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 19. Aug. 2007 <br />
|<br />
|<br />
|<br />
|}<br />
<br />
===='''.:: Week 3 ::.'''====<br />
<br />
Little rearrangements of the parts. Planning of the sequences to order them.<br />
<br />
<br />
===='''.:: Week 4 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 27. Aug. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 28. Aug. 2007 <br />
| <br />
|<br />
| <br />
|-<br />
| Wed, 29. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Thu, 30. Aug. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 31. Aug. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 01. Sept. 2007 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 <br />
| <br />
* Transform pbr322, pcyc177 and pck01 and plated them <br />
| Stefan<br />
|-<br />
| Sun, 02. Sept. 2007 <br />
|<br />
* Prepare o/n of pbr322, pcyc177, pck01<br />
| <br />
* o/n of pcyc177, pck01<br />
* the plates of pcyc177 and pck01 are in the fridge<br />
* transformed pbr322 because the culture didn't grow on the plate<br />
| Stefan<br />
|}<br />
<br />
===='''.:: Week 5 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 03. Sept. 2007 <br />
|<br />
* Prepare new competent cells<br />
* Miniprep pcyc177 and pcK01<br />
* prepare new o/n culture of pbr322<br />
* Run agarose gel of Minipreped plasmids<br />
| <br />
* New competent cells prepared, they are now in the -80° Frezzer in the basement, column #17, dark orange box (we have now 30-35 EDTs of competent cells...)<br />
* Minipreped pcyc177 and pck01 (in the -18° freezer, where the antibiotics are)<br />
* pbr322 didn't grow again, so no o/n could be prepared, but we get a culture from Andy on tuesday<br />
* new o/n of pcyc177 and pck01 prepared (3 Falcons each), because we need to have more plasmids<br />
* 2 boxes of blue pipette tips are in the autoclave<br />
* Stefan ran the agarose gel (?) <br />
| Martin<br>Stefan<br />
|-<br />
| Tue, 04. Sept. 2007 <br />
|<br />
* Miniprep pcyc177 and pck01<br />
* cut the prepped plasmids to test if we've got the right ones<br />
* run agarose gel to test the cut and uncut ones <br />
* prepare new o/n of pbr322 (from Andy) <br />
|<br />
* Miniprep of pcyc177 and pck01 (but not yet tested)<br />
* Prepared 3 o/ns of pbr322 (finally ;-) and each 1 o/n of pcyc177 and pck01, just in case there are problems with the miniprep <br />
| Martin<br>Christian<br />
|-<br />
| Wed, 05. Sept. 2007 <br />
| <br />
* Miniprep of pbr322<br />
* Test-Digest of pcyc177 and pck01 and agarose gel...<br />
* Streak out all three plasmids on new plates, so we have them in reserve <br />
|<br />
* New Plate of pbr322.<br />
* Minipreps and Agarose Gels will be done tomorrow <br />
| Martin<br />
|-<br />
| Thu, 06. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01<br />
* Test with agarose gel <br />
| <br />
* Gel of the older plasmids -> plasmid present<br />
| Christian<br />
|-<br />
| Fri, 07. Sept. 2007 <br />
| <br />
* Miniprep of pbr322, pacyc177, pck01 <br />
| <br />
* Plasmids miniprepped<br />
| Martin<br />
|-<br />
| Sat, 08. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 09. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 6 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 10. Sept. 2007 <br />
| <br />
* Miniprep pBR322<br />
* annealing of different MCSs<br />
* Digest of pCK01 with BamHI+AseI<br />
* digest of pACYC177 with BamHI+PstI<br />
* digest of pBR322 with EcoRI+PstI <br />
all digests o/n<br />
| <br />
<br />
Christian<br />
| | <br />
|-<br />
| Tue, 11. Sept. 2007 <br />
|<br />
* Gelextraction of backbones pBR322, pCK01, pACYC digest did NOT work<br />
* 1x ligation of MCS inside backbones o/d, Trafo<br />
* 1x ligation of MCS inside backbones o/n<br />
| <br />
* plate all 3 plasmids for new minipreps<br />
|<br />
Christian<br />
|-<br />
| Wed, 12. Sept. 2007 <br />
|<br />
* Trafo of o/n ligations<br />
* o/n cultures of putative clones <br />
|<br />
|<br />
Christian<br />
|-<br />
| Thu, 13. Sept. 2007 <br />
| <br />
* Minipreps of putative clones pCK01-MCS and pBR322-MCS<br />
* control digests of putative clones<br />
* new o/n cultures of the putative clones of o/n ligations<br />
| <br />
| <br />
Christian<br />
|-<br />
| Fri, 14. Sept. 2007 <br />
| <br />
*separation of control digests of putative clones <br />
|<br />
'''*pBR322-MCS (Tet-selection) clone2 positive''' <br />
|<br />
Christian<br />
|-<br />
| Sat, 15. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 16. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 7 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 17. Sept. 2007 <br />
|<br />
* new digest of pACYC177 with BamHI+PstI o/n<br />
* digest of pACYC177, pBR322 AP<br />
* ligation of 177 and 322AP<br />
| <br />
* digest of pBR322 AP (the concentration of DNA was too low for pacyc177...)<br />
* ligation of pBR322 o/n<br />
* 100 ml o/n culture to MAXIprep pacyc177<br />
* Transformation of pBR322 AP to have it on plates (because andy only miniprepped them)<br />
| <br />
Christian <br> Martin, Raphael<br />
|-<br />
| Tue, 18. Sept. 2007 <br />
|<br />
* different control digests of pBR322-MCS (Tet) (see last week)<br />
* separation of pACYC177 digest<br />
* Test Digests of pck01 with XbaI, SpeI, PstI, Xba/Pst, Xba/Spe (because all of them should be in the plasmid due to the sequence, and if they are it would be crap!!!)<br />
* Transformation of the ligated pbr322 AP (MCS)<br />
* Prep pacyc177<br />
* Digest prepped pacyc177<br />
|<br />
> no DNA on pACYC177 digest-gel, only degradation smear<br> <br><br />
<br />
* Plates of pbr322 AP grew<br />
* No Digest of pck01 worked due to too low DNA concentration... (che cazzo di low copy plasmids !!!!)<br />
* Miniprepped only 20 ml of the pacyc o/n culture with Quiagen Kit, the results were great! We have loads of DNA! (thank god! )<br />
* Digest of pacyc177 with BamHI (45 µl), then precipitated, in the gel was still very much DNA, but there were still 3 bands, so we guess, that it hasn't cut, maybe because the BamHI in the center is very old, perhaps we should Digest it in Höngg again.<br />
* Digest of pacyc177 with PstI o/n (pray that it will work!)<br />
* New o/n cultures of pck01 (to prep it like pacyc177), pbr322 AP (to prep it too, to have something on stock again, if the ligation didn't work), top10 (to make new competent cells)<br />
* test digest of pck01 with notI, but due to the low DNA concentration I don't think it will work. I took glooves, if it now work, then we have caught some DNases in the earlier test digests<br />
| <br />
<br />
Christian <br><br />
Martin<br>Raphael<br />
|-<br />
| Wed, 19. Sept. 2007 <br />
| <br />
* o/n culture of pbr322 AP (MCS), then test digest and see if it is ligated<br />
* Prep of pck01 and test digests (xba, pst, spe, pvuI, notI)<br />
* check the digests of pacyc177 (pst) and pck01 (notI)<br />
* design new linkers for pck01, design primers for PCR for the extraction of SpeI from pck01 <br />
|<br />
|<br />
|-<br />
| Thu, 20. Sept. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 21. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 22. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 23. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 8 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 24. Sept. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 25. Sept. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 26. Sept. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 27. Sept. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 28. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 29. Sept. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 30. Sept. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 9 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 1. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 2. Oct. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 3. Oct. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 4. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 5. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 6. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 7. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}<br />
<br />
===='''.:: Week 10 ::.'''====<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"<br />
! Date <br />
! TODO's <br />
! Completed <br />
! People<br />
|-<br />
| Mon, 8. Oct. 2007 <br />
|<br />
| <br />
| <br />
|-<br />
| Tue, 9. Oct. 2007 <br />
|<br />
|<br />
| <br />
|-<br />
| Wed, 10. Oct. 2007 <br />
| <br />
|<br />
|<br />
|-<br />
| Thu, 11. Oct. 2007 <br />
| <br />
| <br />
| <br />
|-<br />
| Fri, 12. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sat, 13. Oct. 2007 <br />
| <br />
| <br />
|<br />
|-<br />
| Sun, 14. Oct. 2007 <br />
|<br />
| <br />
|<br />
|}</div>Kdikaiouhttp://2007.igem.org/wiki/index.php/ETHZ/BiologyETHZ/Biology2007-10-15T14:55:07Z<p>Kdikaiou: </p>
<hr />
<div><center>[[Image:Eth_zh_logo_4.png|900px]]</center><br />
<br />
<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | Biology Pespective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Engineering | Engineering Perspective]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br><br />
<br />
<center><font size = '+1'><b> .:: EducatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br><br />
<br />
<p>In this page, you can find an analysis of the function of our system and its relation to epigenetics, its biological design and a list of the parts that it consists of. Are you interested in constructing EducatETH <i>E.coli</i> in your lab? Then under [https://2007.igem.org/ETHZ/Biology/Lab In the Lab], you can find the ingredients and equipment we used, the electronic version of our lab notebook and a presentation of all the difficulties that we encountered. If you are also interested on how EducatETH E.coli was simulated outside the lab, please visit the [[ETHZ/Engineering | Engineering Perspective]]. </p><br><br />
__TOC__<br />
====.:: Introduction ::.====<br />
<br />
<p> EducatETH <i>E.coli</i> is a system which can distinguish between aTc and IPTG based on a previous training phase conducted with the same chemicals and the help of AHL. It composes of three subsystems: the subsystem of constitutively produced proteins, the learning subsystem and the reporting subsystem. The constitutively produced proteins (lacI, TetR and LuxR) control the learning subsystem. At the core of the latter there exists a modified version of the toggle switch found in [1] with two operator sites, so that it only changes its state when both one of the two chemicals (aTc/IPTG) and AHL are present. As AHL is only present during the training phase, the toggle maintains its state during testing, and thus can “memorize”. In the reporting subsystem, four reporters allow supervision of both the chemical the system was trained with and of if the system recognizes the chemical it is being exposed to in the testing phase as one it has been trained with or not.</p><br><br />
<br />
====.:: The whole system ::.====<br />
<br />
<p>The biological design of EducatETH <i>E.coli</i> is presented on [[Image:new_learning_system3.png|thumb|left|300px|EducatETH System '''(Fig. 1)''']] . In the following, we will clarify the function of all depicted components. (Are you interested in how the complex system of Fig. 1 was modelled? Then visit the [[ETHZ/Engineering | Engineering Perspective]]!)</p><br><br />
<br />
=====.:: Constitutive subsystem ::.=====<br />
<br />
<p>The constitutively produced proteins of the system are LacI, TetR and LuxR. The LuxR part has a special function: when AHL is present, it forms a LuxR-AHL complex which acts on the learning subsystem (more on this later). At the moment, we will consider that AHL is absent and therefore LuxR cannot act on any subsystems. The TetR and lacI parts behave similarly: more specifically, the tetR protein in the absence of aTc inhibits the production of p22cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the p22cII production is no longer inhibited (and thus aTc is produced). Respectively, cI is produced when IPTG is present.</p><br />
<br />
=====.:: Learning subsystem ::.=====<br />
<br />
<p>The learning subsystem is a toggle switch with two operator sites. The upper part of the toggle (cI production) has operator sites for the LuxR-AHL complex and p22cII (which has been in turn induced by aTc). The LuxR-AHL complex induces the cI production, whereas p22cII inhibits it. The lower part of the toggle (p22cII production) has operator sites for the LuxR-AHL comple and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the p22cII production and cI inhibits it. Therefore, the switch always requires the presence of the LuxR-AHL complex in order for it to operate. Its state depends on the presence of p22cII and cI into the system, which in curse was caused through the exposure of the system to aTc and IPTG.</p><br />
<br />
=====.:: Reporting subsystem ::.=====<br />
<br />
<p>There are four reporters in the system. CFP and YFP are active during the training phase of the system and show which chemical the system is exposed to during training, whereas GFP and RFP are active during the testing phase and show if the system is exposed to the same chemical as in training or not. <br />
More specifically, the YFP protein production is regulated with help of two operator sites controlled by cI and aTc. cI inhibits YFP production and aTc induces it. Therefore, YFP is produced when the system is exposed to aTc. In a similar manner, the CFP production is produced when the system is exposed to IPTG. <br />
The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br><br />
<br />
====.:: System phases ::.====<br />
<br />
<p>The system operation is divided into two main phases: the training phase and the testing phase. The training phase itself is also subdivided into two phases: seeing and memorizing. During seeing, the system is first exposed to one of the two chemicals it is designed to recognize (aTc and IPTG). AHL is then added and the system’s internal toggle switch reaches a steady state. During memorizing, the chemical used during seeing is removed and only AHL is retained. This maintains the toggle switch to its acquired steady state, which is reported with YFP (if aTc was seen) or CFP (if IPTG was seen). During the testing phase, the system is exposed to any of the two chemicals (aTc or IPTG), but not to AHL. By comparing its toggle switch state with the effect of the newly introduced chemical, the system shows a different response if it has previously been exposed to this chemical (GFP) or not (RFP).The following table presents all possible paths that may be taken by the system during all phases of operation according to the external stimuli. </p><br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
! <br />
! aTc<br />
! IPTG<br />
! AHL<br />
! p22cII<br />
! cI<br />
! Reporting <br />
|- <br />
| '''Start'''<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
| no<br />
|- <br />
| '''Learning'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| '''Seeing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Memorizing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|- <br />
| Trained with aTc<br />
| yes<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yfp<br />
|- <br />
| Trained with IPTG<br />
| no<br />
| yes<br />
| yes<br />
| no<br />
| yes<br />
| cfp<br />
|- <br />
| '''Testing'''<br />
| <br />
| <br />
| <br />
| <br />
| <br />
| <br />
|-<br />
| Trained with aTc<br>Tested with aTc<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| no<br />
| gfp<br />
|- <br />
| Trained with aTc<br>Tested with IPTG<br />
| no<br />
| yes<br />
| no<br />
| ?<br />
| ?<br />
| rfp<br />
|- <br />
| Trained with IPTG<br>Tested with IPTG<br />
| no<br />
| yes<br />
| no<br />
| no<br />
| yes<br />
| gfp<br />
|- <br />
| Trained with IPTG<br>Tested with aTc<br />
| yes<br />
| no<br />
| no<br />
| ?<br />
| ?<br />
| rfp<br />
|- <br />
<br />
|}<br />
<br />
=====.:: Further thoughts on the system phases ::.=====<br />
<br />
TODO: put Stefan's new text here. Text should be smaller.<br />
<br />
<p>[[Image:LearningSystemOverview.jpg|thumb|right|200px| Abstraction of our learning system '''(Fig. 2)''']]<br />
<br />
A straightforward approach on how to describe learning behavior and adaptive evolution, can be see in Figure 1. We can separate the process in two phases: a <i>training</i> or <i>learning phase</i> (shown in blue), and an <i>application</i> or <i>"real world" phase</i> (shown in pink), and describe our system as a multiple state automaton. The examined system can alter its state according to a certain input/stimuli that it was exposed to in the first phase. In this binary example, the system changes its state from <i>a</i> to <i>b</i> when it is exposed to a first training-phase-input (<i>IT1</i>). Similarly, the system changes its state from <i>a</i> to <i>c</i> when the other training-phase-input (<i>IT2</i>) is applied.<br />
<br />
The principle described above is also valid in the <i>application phase</i>. In the application phase, we have two possibilities for initial system state. Thus, the automaton expands with four more states. Depending on which state the system is at the start of the <i>application phase</i>, and which chemical it is exposed to, it reaches a different final state. State <i>d</i> is reached if the first out of two possible application-phase-inputs (<i>IA1</i>) is applied while the system is at state <i>b</i>. In the end, we can differentiate between the possibilities of the system being trained with one chemical, and being exposed to a different chemical in the application phase. Since we have two different <i>training</i> chemicals, and two different <i>application</i> chemicals, we reach the final number of four states.<br />
<br />
In the following, we elaborate on the two phases of our system:<br />
<ul><br />
<li>How can we describe learning ability with this approach?<br>Define the training-phase-inputs themselves as the information entities to be learned. This implies that after the training phase, the information is permanently stored in the system - ''a memory has been created''. According to its memory, the system will behave differently when it is exposed to a certain stimuli in a later stage.<br />
<br />
<li>If one have a look at ''Figure 1'' once more, one can easily spot similarity to family trees or [http://en.wikipedia.org/wiki/Phylogenetic_tree phylogenetic trees]. This raises the concept of divergent evolution (adaptation). There are several differences to the learning model: First of all, the states don't describe a single living entity with changing characteristics but different populations or species. Secondly, the training-phase-inputs and application-phase-inputs are not related to information inputs but rather to events acting on those populations/species. Thirdly, there is no specific training- and application phase but just two phases with different events/stimuli acting on the populations/species.<br>The following example might show this concept more clearly: Let's say the population of precursor (= ancestor) species ''a'' is splitted into two subpopulations (''a1'' and ''a2'') due to emigration of subpopulation ''a1'' to another geographic region. Application-phase-input 1 (''AI1'') would then equal "emigration" (''a'' -> ''a2'') and application-phase-input 2 (''AI2'') would be "no emigration" (''a'' -> ''a1''). The isolated populations then undergo changes as they (1.) become subjected to dissimilar selective pressures or (2.) they independently undergo genetic drift. When the populations come back into contact, they have evolved such that they are reproductively isolated and are no longer capable of exchanging genes [1]. Therefore, subpopulation ''a1'' has evolved into species ''b'', whereas subpopulation ''a2'' has evolved into species ''c''.<br>This example explains the principle of [http://en.wikipedia.org/wiki/Allopatric_speciation allopatric speciation]. However, our model is not limited to this: Peripatric speciation, parapatric speciation, sympatric speciation or artificial speciation can also be expressed through this model system. Another highly exciting aspect of our model system is, that it can describe '''Evolution without changing the DNA content over time!''' How this can be achieved is explained in more detail in the following sections.<br />
<li>(The automaton of Fig. 1 presents similarities to family trees, or [http://en.wikipedia.org/wiki/Phylogenetic_tree phylogenetic trees]. According to different external stimuli, the initial population divides, and evolves into different species. The proposed automaton model can be used to explain the concepts of peripatric speciation, parapatric speciation, sympatric speciation or artificial speciation e.t.c. However, the most imporant fact, and what has stimulated our research in the area, is, to <i>create a biological system that can evolve without changing its DNA content over time</i>).<br />
</ul></p><br />
<br />
====.:: System parts ::.====<br />
<br />
EducatETH E.coli consists of 11 parts which may be synthesized independently. Please note that four of them (4,5 and 8,9) form together two functional system units. They have been separated to ensure comparable part lengths and thus enable easier introduction into plasmids.<br />
<br />
The system consists of 11 parts that can be synthesized independently (want to know how this is done in the lab? Then visit our [https://2007.igem.org/ETHZ/Biology/Lab In the Lab] page!)<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Single System parts'''<br />
|-<br />
|-<br />
! 1<br />
| TetR production <br />
| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739001 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| LacI production <br />
| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739002 SpecifiedComponents</partinfo><br />
|-<br />
! 3<br />
| LuxR production <br />
| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]<br />
| constitutive subsystem<br />
| <partinfo>BBa_I739003 SpecifiedComponents</partinfo><br />
|-<br />
! 4<br />
| 1st half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739004 SpecifiedComponents</partinfo><br />
|-<br />
! 5<br />
| 2nd half of P22 cII / EYFP production<br />
| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005 ]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739005 SpecifiedComponents</partinfo><br />
|-<br />
!6<br />
| CI production<br />
|<br />
| learning subsystem<br />
|-<br />
! 7<br />
| P22 cII production<br />
| <br />
| learning subsystem<br />
|-<br />
! 8<br />
| 1st half of cI / ECFP production<br />
|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739008 SpecifiedComponents</partinfo><br />
|-<br />
! 9<br />
| 2nd half of cI / ECFP production<br />
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009] <br />
| reporting subsystem<br />
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo><br />
|-<br />
! 10<br />
| RFP production <br />
| <br />
| reporting subsystem<br />
|-<br />
! 11<br />
| GFP production <br />
| <br />
| reporting subsystem<br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Double Promoters'''<br />
|-<br />
|-<br />
! 1<br />
| cI negative / TetR negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739102 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| lacI negative / P22 cII negative promoter<br />
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]<br />
| reporting subsystem<br />
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo><br />
|}<br />
<br />
{| class="wikitable" border="1" cellspacing="0" cellpadding="2" style="text-align:center; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;" <br />
|+ '''Proof of Concept'''<br />
|-<br />
|-<br />
! 1<br />
| PoC promoter<br />
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739101 SpecifiedComponents</partinfo><br />
|-<br />
! 2<br />
| PoC intermediate<br />
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]<br />
| proof of concept, no part of the system <br />
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo><br />
|}<br />
<br><br />
<br />
<br />
====.:: References ::.====<br />
<p>[1] <i>Standard Assembly Process</i>, http://partsregistry.org/Assembly:Standard_assembly</p><br><br />
<br />
====.:: To Do ::.====<br />
<br />
=====.:: New ::.=====<br />
<p><ul><br />
<li> Update and correct parts in parts list. Write better in a table<br />
<li> Update and correct full system scheme<br />
<li> Update graph scheme (made by Stefan) using aTc, IPTG instead of it1,2 and ia1,2<br />
<li> Which reporters are active when? I think CFP and YFP are not active only during training. Change text if needed.<br />
<li> Proposed terminology: seeing, memorizing<br />
<li> What are GFP, RFP controlled by? Is the full system scheme correct there?<br />
<li> What are the “double promoters” mentioned?<br />
<li> Check my terminology (operator sites etc)<br />
<li> Put Stefan's updated part on epigenetics<br />
<li> How was Sven’s standard notation on how to write differently proteins, dna, rna?<br />
<li> Fill in table completely, make it more reading-friendly<br />
<li> Make "In the lab page", replace links.<br />
<li> Put image with 11 system parts (updated one, created by katerina)<br />
</ul></p><br><br />
=====.:: Old::.=====<br />
<p><ul><br />
<li>''Katerina'': 1. Number system parts on both figures for easier reference.<br />
<li>''Katerina'': 2. Add more info on all system parts and link to the ones existing in the registry. Write info on the ones that didn't exist in the registry (with detailed info such as addition of bp's as Christian and Sven had done).<br />
<li>''Katerina'': 3. Add cloning plan. (Christos: Maybe the details will be at the team note's?)<br>Martin: I wouldn't put it here, I mean the cloning plan is really nothing special, it's like an auxiliary calculation for a polynom division... Not exciting and everyone could do it... Please correct me if I'm wrong. By the way I wouldn't write so much about the methods with which we cloned the parts in. These are generally known and every biologist knows them. So I think a jury member who knows all the stuff (which he really should) will be bored if he has to work through the whole assembly and plasmid stuff. I would just say, that we use three Plasmids (with names) for insertion of our system into the bacteria and I would also write which part is on which plasmid, but not more. I would more concentrate on the System, how it's working, what parts we've used and so on....<br />
<li>''Christos'': 1. The picture with the abstraction - maybe we can put better names on the arrows.<br />
<li>''Christos'': 2. We can make figure 1 more specific. Instead of having IT and IA, we can have Chemical 1, Chemical 2, and then again. I think it will be more obvious like that.<br />
<li>''Christos'': 3. Nice descriptions Stefan. However, I made the text less, and kept the original idea, since I felt like it was moving away from the purpose, which is a simple introduction and clarification of concepts. I also removed the pictures. I have backups of everything, so, we can put it back if the others disagree.<br />
<li>''Christos'': 4. Figure 3 needs to be replaced with the new parts.<br />
<li>''Christos'': 5. Are we sure the plasmids that we say are the correct ones? Sven said they were changed, but I didn't really get it.<br>Martin: Yes, they are the right ones... If not, the shit is hitting the fan - albeit in my opinion it has already hit the fan, but not due to the plasmids more due to Genefart...I can really assure you, that they are right. ;-)<br />
</ul></p><br></div>Kdikaiou