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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#The_Complete_System">The Complete System</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team#Team_Description">Team Description</a>
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<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>
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<center><font size = '+2'><b> .:: ETH Zurich - EducatETH ''E.coli'' ::. </b></font></center><br>
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<p align="center"><font size="6"><b>ETH Zurich - educatETH <i>E.coli</i> System</b></font></p>
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=='''.:: Introduction ::.'''==
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=Introduction=
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<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> 
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CHANGE THIS: HERE COMES THE MOTIVATION WE ARE DOING A COOL PROJECT!!!
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... 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 ...
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<p>It is the third 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> ! More specifically, in our project (Fig. 1)  [[Image:Educateth_Ecoli.png|thumb|The ETH Zurich team undertook the task of engineering a learning mechanism for <i>E.coli</i>. '''(Fig. 1)'''|500px]] 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>
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<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>
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==Motivation==
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====='''.:: Team Members ::.'''=====
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[[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]]
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KICK PEOPLE TO HAVE A PROPER LINK
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<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>
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<li><i>Project advisors</i>: [https://2007.igem.org/User:sven Sven Panke], Joerg Stelling
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<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/User:LiNan Nan Li], [https://2007.igem.org/User:Stefan Stefan Luzi]
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<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]
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</ul>
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<p>For more information on the team members, follow the links or visit [[ETHZ/Meet_the_team | Meet the Team]].</p>
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====='''.:: Motivation ::.'''=====
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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.  
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<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 big 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>
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In other words, in this project '''we are educating the <i>E.coli</i>'''!
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====='''.:: <i>E.coli</i> Intelligence (E.I) - Specifications and analogies ::.'''=====
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===Multipurpose Cell Lines===
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<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>
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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.  
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<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>
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This means that one applies an “input engineering” instead of a “DNA engineering” approach. If one extends this idea to several multi-inducible toggle switches being harbored in the same cell line, the number of possible phenotypes increases to 2<i><sup>n</sup></i>, where <i>n</i> 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.
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(Want to read more about this? Visit the [[ETHZ/Engineering| Engineering Perspective]]!)</p>
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<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>
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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.
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(Want to see the biological design of our modified toggle? Visit the [[ETHZ/Biology | Biology Perspective]]!)</p>
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====='''.:: A short system description ::.'''=====
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===Link to Epigenetics===
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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.
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<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).
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===Intelligent Biosensors and Self-Adaptation===
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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>
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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.
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{| 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;"                   
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==Team Members==
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! Training <br>chemical             
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[[Image:ETHZ_Group_photo_6.png|right|thumb|The ETH Zurich iGEM2007 Team|270px]]
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! Testing <br>chemical             
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! Fluorescence <br>during training
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! Fluorescence <br> during testing
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|-   
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| aTc
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| aTc
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| CFP or YFP?
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| GFP
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|-     
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| aTc                   
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| IPTG
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| CFP or YFP?
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| RFP
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|-   
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| IPTG                   
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| aTc                     
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| CFP or YFP?
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| RFP
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|-       
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| IPTG
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| IPTG
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| CFP or YFP?
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| GFP
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|}
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====='''.:: References ::.'''=====
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The ETH Zurich team consists of a good mixture between biologists and engineering students.  
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[1]: Toggle Switch
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We are:
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<ul>
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<li><i>Undergraduate students</i>: <br>
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[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>
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[https://2007.igem.org/Nan_Li Nan Li],
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[https://2007.igem.org/User:Stefan Stefan Luzi]
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<li><i>Graduate students</i>: <br>
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[http://christos.bergeles.net Christos Bergeles],
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[http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm], <br>
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[http://www.fussenegger.ethz.ch/people/kemmerc Christian Kemmer],
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[https://2007.igem.org/User:JoeKnight Joseph Knight], <br>
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[http://www.ricomoeckel.de Rico Möckel],
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[http://csb.inf.ethz.ch/people/uhr.html Markus Uhr]
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<li><i>Project advisors</i>: <br>
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[http://www.ipe.ethz.ch/laboratories/bpl/people/panke Sven Panke], <br>
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[http://csb.inf.ethz.ch/people/stelling.html Joerg Stelling]
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</ul>
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====='''.:: Acknowledgements ::.'''=====
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For more information about us, visit our [[ETHZ/Meet_the_team | Meet the Team]] page.
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<ul>
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<li><p>[http://europa.eu The European Union]</p>
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==Acknowledgments==
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<li><p>[http://www.ethz.ch The ETH Zurich]</p>
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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.
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<li><p>[http://www.geneart.com GENEART]</p>
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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]:
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====='''.:: Links ::.'''=====
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<center>
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<ul>
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{| border="0"
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<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]</p>
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|-
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<li><p>[https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]</p></ul><br>
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| [http://europa.eu http://www.tik.ee.ethz.ch/~thohm/EU.gif]
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| width="40" |
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| [http://www.ethz.ch http://www.tik.ee.ethz.ch/~thohm/ethlogo.jpg]
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| width="40" |
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| [http://www.geneart.com http://www.tik.ee.ethz.ch/~thohm/geneart.gif]
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|}
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</center>
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==Site Map==
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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.
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The site map of our wiki is the following:
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{| 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;"
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!width="34%"| Modeling Pages                       
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!width="22%"| Biology Pages
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!width="22%"| ETHZ Team Pages
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!width="22%"| Links
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|-
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| [[ETHZ/Model | Modeling of the learning system]]
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| [[ETHZ/Biology | Biological implementation]]
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| [[ETHZ/Meet_the_team | Team page]]
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| [https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]
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| [[ETHZ/FlipFlop | Representation using flip-flops]]
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| [[ETHZ/Biology/parts | Biobricks/parts]]
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| [[ETHZ/Pictures | Pictures]]
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| [https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]
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|-
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| [[ETHZ/FSM | Representation using finite state machines]]
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| [[ETHZ/Biology/Lab | Lab notes]]
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| [[ETHZ/Internal | Brainstorming sessions]]
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| [[ETHZ/Simulation | Model simulations and theoretical considerations]]
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| [[ETHZ/Parameters | Parameters used in our simulations]]
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== References ==
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=='''.:: To Do ::.'''==
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[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 />
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====='''.:: New ones ::.'''=====
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[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 />
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<p><ul>
 
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<li> Change banner on top of page. Stefan: Can we please change the banner back to the old version?!? It was MUCH nicer.
 
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<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.
 
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<li> Possibly put table of contents.
 
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<li> Possibly take EducatETH <i>E.coli</i> from Figure.
 
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<li> Change Figure 2.
 
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<li> Possibly change figure caption format from xxx (Fig. x) to Fig.x: xxx. Put second format on Figure 2 for all to check.
 
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<li> Fix table showing possible system states and reporters. Maybe with some color?
 
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<li> Put cross-reference on table.
 
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<li> Align table left
 
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<li> Add which reporters do what in paragraph.
 
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<li> Put (compressed!) team photo instead of Fig.1
 
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<li> Fix paragraph with system description, looks somehow bulky. Maybe italics or bold on important things?
 
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<li> Fix reference on toggle switch.
 
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<li> Possibly remove Introduction heading altogether, and just start with the text.
 
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</ul></p>
 
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====='''.:: Old ones ::.'''=====
 
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<p><ul>
 
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<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'')
 
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<li>Need picture: Einstein ecoli (''Sylke: does Stefan have the layer file?'')
 
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<li>Stupidity: All E. Colis are equal, but our E. Colis are more equal than the others :D
 
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<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.
 
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<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)
 
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</ul></p>
 
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Latest revision as of 20:20, 26 October 2007

ETHZ banner.png

 


ETH Zurich - educatETH E.coli System

Introduction

"All E.coli 's are equal, but some E.coli 's are more equal than others..." (freely adapted from "Animal Farm" by George Orwell)

... this is what George Orwell would have written, were he a synthetic biologist. In the E.coli colonies on petri dishes, all bacteria are equal; except for some special ones. Our project is about designing such special E.coli 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 ...

Motivation

Fig. 1: 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)

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. In other words, in this project we are educating the E.coli!

Multipurpose Cell Lines

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.

This means that one applies an “input engineering” instead of a “DNA engineering” approach. If one extends this idea to several multi-inducible toggle switches being harbored in the same cell line, the number of possible phenotypes increases to 2n, 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.

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 double promoters to the Registry of Standard Biological Parts, which can be helpful for engineering systems which exhibit a desired behavior only at specific times.

Link to Epigenetics

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.

Intelligent Biosensors and Self-Adaptation

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.

Team Members

The ETH Zurich iGEM2007 Team

The ETH Zurich team consists of a good mixture between biologists and engineering students. We are:

For more information about us, visit our Meet the Team page.

Acknowledgments

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 Sven Panke's Lab, especially Andreas Meyer who was always there for us when we had a problem. Additionally, we would like to thank Alfredo Franco-Obregón's lab and Oralea Büchi for the help with the flow cytometry.

We would also like to acknowledge the financial support by EU, the ETH Zurich, and GeneArt:

http://www.tik.ee.ethz.ch/~thohm/EU.gif http://www.tik.ee.ethz.ch/~thohm/ethlogo.jpg http://www.tik.ee.ethz.ch/~thohm/geneart.gif

Site Map

In this wiki, we will present to you a detailed description of the proposed system: starting with the modeling of the system, we describe both, simulations and theoretical considerations of the system, as well as the actual implementation using bio-bricks accompanied by our lab notes. Additionally, you will find further information on the team, more details about ideas we developed before we came up with the system we finally implemented, and some pictures documenting our work.

The site map of our wiki is the following:

Modeling Pages Biology Pages ETHZ Team Pages Links
Modeling of the learning system Biological implementation Team page The ETH Zurich 2005 project
Representation using flip-flops Biobricks/parts Pictures The ETH Zurich 2006 project
Representation using finite state machines Lab notes Brainstorming sessions
Model simulations and theoretical considerations
Parameters used in our simulations

References

[1] Bird A "Perceptions of epigenetics", Nature 447:396-398, 2007
[2] Ptashne M "On the use of the word ‘epigenetic’", Current Biology 17(7):R233-R236, 2007


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