ETHZ/Biology

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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><br>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Introduction">Introduction</a>
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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>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#Introduction">Introduction</a>
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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/Biology#System_Phases">System Phases</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#Current_Cloning_Status">Current Cloning Status</a>
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<center><font size = '+1'><b> .:: educatETH <i>E.coli</i> - Biology Perspective ::. </b></font></center><br>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team#The_ETH_Zurich_07_Team">The ETH Zurich 07 Team</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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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Internal">Brainstorming Page</a>
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<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 modeled and simulated outside the lab, please visit the [[ETHZ/Model | modeling]] and [[ETHZ/Simulation | simulations]] web pages. </p><br>
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== Introduction ==
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= Introduction =
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<p> 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 training phase conducted with the same chemicals and the help of [http://partsregistry.org/Acyl-HSLs 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 and extended version of the Elowitz toggle switch found in [1]. 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 training phase, the toggle maintains its state during testing, 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 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>
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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.
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== The complete system ==
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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.
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<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>
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== The Complete System ==
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=== Constitutive subsystem ===
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<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>
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<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 P22 cII and LacI in the absence of IPTG inhibits the production of cI. When aTc is present, however, the P22 cII production is no longer inhibited (and thus p22 cII is produced). Respectively, cI is produced when IPTG is present.</p>
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==== Constitutive Subsystem ====
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=== Learning subsystem ===
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<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>
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<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 P22 cII (which has in turn been induced by aTc). The LuxR-AHL complex induces the cI production, whereas P22 cII inhibits it. The lower part of the toggle (P22 cII production) has operator sites for the LuxR-AHL complex and cI (which has been induced by IPTG). Similarly with the upper part, the LuxR-AHL complex induces the P22 cII 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 P22 cII and cI into the system, which in return was caused through the exposure of the system to aTc and IPTG.</p>
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==== Learning Subsystem ====
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=== Reporting subsystem ===
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<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>
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<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 training phase of the system and show which chemical the system is exposed to during training, whereas all four reporters (the latter and 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.
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==== Reporting Subsystem ====
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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.
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The GFP production is regulated with help of two operator sites controlled by lacI and .</p><br>
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== System phases ==
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<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.
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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:
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*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).
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*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).
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*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).
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*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>
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<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>
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This behaviour is visualized in Fig. 2.
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[[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]]
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== System Phases ==
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<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>
{| 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;"         
{| 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;"         
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  | no
  | no
  | no
  | no
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  | non
  |-       
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  | colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''
  | colspan="7" style="background:#96c9cf;" align="center"| '''Learning'''
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  | yes
  | yes
  | no
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  | YFP (fading)<br>finally no color
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  | Trained with IPTG
  | Trained with IPTG
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  | no
  | no
  | yes
  | yes
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  | CFP
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  | CFP (fading)<br>finally no color
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  | colspan="7" style="background:#96c9cf;" align="center"|  '''Recognition'''
  | colspan="7" style="background:#96c9cf;" align="center"|  '''Recognition'''
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  |}
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=== Further thoughts on the system ===
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== System Parts ==
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==== Link to Epigenetics ====
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<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>
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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 inducer molecules (ATc or IPTG). E.g. if ATc is added, one subpopulation will be red, while the other will be yellow. Therefore the epigenetic feature here is the binding of specific repressor proteins, whose production is dependend on the toggle switch state, to their corresponding operator sequences.
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== Current Cloning Status (26.10.07) ==
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== System parts ==
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We have sent 9 new parts and 3 new plasmids to the registry. As soon as the missing parts are available in their destined plasmids, we will forward them to the parts registry as well. Additionally, we will be able to test parts of our system next week, using the FACS machine provided by [http://www.facs.ethz.ch Alfredo Franco-Obregón's lab].
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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.
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{| 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;"   
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|+ '''Single System Parts'''
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!width="111"| 1
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|width="285"| TetR production
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| [http://partsregistry.org/Part:BBa_I739001 BBa_I739001]
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|width="250"| constitutive subsystem
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|width="157"| <partinfo>BBa_I739001 SpecifiedComponents</partinfo>
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! 2
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| LacI production
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| [http://partsregistry.org/Part:BBa_I739002 BBa_I739002]
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| constitutive subsystem
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| <partinfo>BBa_I739002 SpecifiedComponents</partinfo>
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|-
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! 3
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| LuxR production
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| [http://partsregistry.org/Part:BBa_I739003 BBa_I739003]
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| constitutive subsystem
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| <partinfo>BBa_I739003 SpecifiedComponents</partinfo>
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! 4
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| 1st half of P22 cII / EYFP production
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| [http://partsregistry.org/Part:BBa_I739004 BBa_I739004]
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| reporting subsystem
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| <partinfo>BBa_I739004 SpecifiedComponents</partinfo>
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! 5
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| 2nd half of P22 cII / EYFP production
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| [http://partsregistry.org/Part:BBa_I739005 BBa_I739005]
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| reporting subsystem
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| <partinfo>BBa_I739005 SpecifiedComponents</partinfo>
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|-
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!6
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| cI production
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| [http://partsregistry.org/Part:BBa_I739006 BBa_I739006]
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| learning subsystem
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| <partinfo>BBa_I739006 SpecifiedComponents</partinfo>
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! 7
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| P22 cII production
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| [http://partsregistry.org/Part:BBa_I739007 BBa_I739007]
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| learning subsystem
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| <partinfo>BBa_I739007 SpecifiedComponents</partinfo>
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! 8
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| 1st half of cI / ECFP production
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|[http://partsregistry.org/Part:BBa_I739008 BBa_I739008]
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| reporting subsystem
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| <partinfo>BBa_I739008 SpecifiedComponents</partinfo>
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! 9
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| 2nd half of cI / ECFP production
+
-
| [http://partsregistry.org/Part:BBa_I739009 BBa_I739009]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739009 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 10
+
-
| RFP production
+
-
| [http://partsregistry.org/Part:BBa_I739010 BBa_I739010]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739010 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 11
+
-
| GFP production
+
-
| [http://partsregistry.org/Part:BBa_I739011 BBa_I739011]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739011 SpecifiedComponents</partinfo>
+
-
|}
+
-
 
+
-
{| 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;"   
+
-
|+ '''Composite System Parts'''
+
-
|-
+
-
|-
+
-
!width="111"| 1+2+3
+
-
|width="285"| tetR + lacI + luxR production
+
-
| [http://partsregistry.org/Part:BBa_I739013 BBa_I739013]
+
-
|width="250"| constitutive subsystem
+
-
|width="157"| <partinfo>BBa_I739013 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 4+5
+
-
| P22 cII + EYFP production
+
-
| [http://partsregistry.org/Part:BBa_I739015 BBa_I739015]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739015 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 8+9
+
-
| cI + ECFP production
+
-
| [http://partsregistry.org/Part:BBa_I739016 BBa_I739016]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739016 SpecifiedComponents</partinfo>
+
-
|-
+
-
! (4+5)+(8+9)
+
-
| (P22 cII + EYFP) + (cI + ECFP) production
+
-
| [http://partsregistry.org/Part:BBa_I739017 BBa_I739017]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739017 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 6+7
+
-
| cI + P22 cII production
+
-
| [http://partsregistry.org/Part:BBa_I739018 BBa_I739018]
+
-
| learning subsystem
+
-
| <partinfo>BBa_I739018 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 10+11
+
-
| RFP + GFP production
+
-
| [http://partsregistry.org/Part:BBa_I739019 BBa_I739019]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739019 SpecifiedComponents</partinfo>
+
-
|-
+
-
! (6+7)+(10+11)
+
-
| (cI + P22 cII) + (RFP + GFP) production
+
-
| [http://partsregistry.org/Part:BBa_I739020 BBa_I739020]
+
-
| learning/reporting subsystem
+
-
| <partinfo>BBa_I739020 SpecifiedComponents</partinfo>
+
-
|}
+
-
 
+
-
 
+
-
{| 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;"     
+
-
|+ '''Double Promoters'''
+
-
|-
+
-
|-
+
-
!width="111"| 1'
+
-
|width="285"| cI negative / tetR negative promoter
+
-
| [http://partsregistry.org/Part:BBa_I739102 BBa_I739102]
+
-
|width="250"| reporting subsystem
+
-
|width="157"| <partinfo>BBa_I739102 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 2'
+
-
| lacI negative / P22 cII negative promoter
+
-
| [http://partsregistry.org/Part:BBa_I739103 BBa_I739103]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739103 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 3'
+
-
| luxR/HSL positive / P22 cII negative promoter
+
-
| [http://partsregistry.org/Part:BBa_I739104 BBa_I739104]
+
-
| learning subsystem
+
-
| <partinfo>BBa_I739104 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 4'
+
-
| luxR/HSL positive / cI negative promoter
+
-
| [http://partsregistry.org/Part:BBa_I739105 BBa_I739105]
+
-
| learning subsystem
+
-
| <partinfo>BBa_I739105 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 5'
+
-
| tetR negative / P22 cII negative promoter
+
-
| [http://partsregistry.org/Part:BBa_I739106 BBa_I739106]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739106 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 6'
+
-
| cI negative / lacI negative promoter
+
-
| [http://partsregistry.org/Part:BBa_I739107 BBa_I739107]
+
-
| reporting subsystem
+
-
| <partinfo>BBa_I739107 SpecifiedComponents</partinfo>
+
-
|}
+
-
 
+
-
{| 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;"     
+
-
|+ '''Proof of Concept'''
+
-
|-
+
-
|-
+
-
!width="111"| 1"
+
-
|width="285"| PoC promoter
+
-
| [http://partsregistry.org/Part:BBa_I739101 BBa_I739101]
+
-
|width="250"| proof of concept, no part of the system
+
-
|width="157"| <partinfo>BBa_I739101 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 2"
+
-
| PoC intermediate
+
-
| [http://partsregistry.org/Part:BBa_I739014 BBa_I739014]
+
-
| proof of concept, no part of the system
+
-
| <partinfo>BBa_I739014 SpecifiedComponents</partinfo>
+
-
|-
+
-
! 3"
+
-
| PoC composite
+
-
| [http://partsregistry.org/Part:BBa_I739021 BBa_I739021]
+
-
| proof of concept, no part of the system
+
-
| <partinfo>BBa_I739021 SpecifiedComponents</partinfo>
+
-
|}
+
-
<br>
+
== References ==
== References ==
-
<p>
 
-
[http://www.nature.com/nature/journal/v403/n6767/abs/403335a0.html &#91;1&#93; Elowitz MB and Leibler S] <i>"A synthetic oscillatory network of transcriptional regulators"</i>, Nature 403:335-338, 2000<br />
 
-
[http://www.nature.com/nature/journal/v447/n7143/abs/nature05913.html;jsessionid=62903C604764B175945C03DB8639ECBD &#91;2&#93; Bird A] <i>"Perceptions of epigenetics"</i>, Nature 447:396-398, 2007 <br />
 
-
[http://linkinghub.elsevier.com/retrieve/pii/S096098220701007X &#91;3&#93; Ptashne M] <i>"On the use of the word ‘epigenetic’"</i>, Current Biology 17(7):R233-R236, 2007<br />
 
-
 
-
== To Do ==
 
-
=== New ===
+
[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 />
-
<p><ul>
+
-
<li> <span style=color:blue;>Update and correct parts in parts list. Write better in a table</span>
+
-
<li> <span style=color:blue;>Update and correct full system scheme</span>
+
-
<li> <span style=color:red;>What is the proof of concept mentioned?</span>
+
-
<li> <span style=color:red;>Are you sure about the reporters? This is not what I understood. I though YFP and CFP were also during testing. Yes! I'm still updating the text :-)</span>
+
-
<li> <span style=color:red;>I don't think it is wise to make p22cII in P22 cII, since we have in the first way all over the place. Stefan: Yes I know :-( However we have different descriptions: p22cII, cII, P22CII, p22 cII, P22 cII. However, P22 cII is the correct one. I'm not sure how we can solve this (minor!) problem.</span>
+
-
<li> <span style=color:red;>Try to improve table with system phases. It doesn't look so nice...</span>
+
-
<li> <span style=color:red;>In the memorizing phase, is there color or not? - Nope, there is no color - There are colors, however they will disappear within 2-3 hours.</span>
+
-
<li> <span style=color:red;>We need to put the complete spaghetti system as well.</span>
+
-
<li> Check my terminology (operator sites etc)
+
-
<li> Put Stefan's updated part on epigenetics
+
-
<li> Fill in table completely, make it more reading-friendly
+
-
</ul></p><br>
+

Latest revision as of 23:14, 26 October 2007

ETHZ banner.png

 


Introduction

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 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.

educatETH E.coli 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.

The Complete System

Fig. 1: Gene interaction network of educatETH E.coli
The biological design of educatETH E.coli 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 System Modeling!)

Constitutive Subsystem

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.

Learning Subsystem

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.

Reporting Subsystem

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. 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:

  • 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).
  • 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).
  • 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).
  • 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).

This behaviour is visualized in Fig. 2.
Fig. 2: 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.

System Phases

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:

System phases
aTc IPTG AHL p22cII cI Reporting
Start
no input no no no no no non
Learning
Trained with aTc yes no no yes no YFP
Trained with IPTG no yes no no yes CFP
Memorizing
Trained with aTc yes no yes yes no YFP (fading)
finally no color
Trained with IPTG no yes yes no yes CFP (fading)
finally no color
Recognition
Trained with aTc
Tested with aTc
yes no yes yes no YFP
Trained with aTc
Tested with IPTG
no yes yes yes no GFP
Trained with IPTG
Tested with IPTG
no yes yes no yes CFP
Trained with IPTG
Tested with aTc
yes no yes no yes RFP

System Parts

educatETH E.coli was implemented with 11 basic parts designed by the ETH Zurich team. 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.
(Are you interested in this information because you want to implement educatETH E.coli in your lab? Then visit our In the Lab page!)

Current Cloning Status (26.10.07)

We have sent 9 new parts and 3 new plasmids to the registry. As soon as the missing parts are available in their destined plasmids, we will forward them to the parts registry as well. Additionally, we will be able to test parts of our system next week, using the FACS machine provided by [http://www.facs.ethz.ch Alfredo Franco-Obregón's lab].

References

[http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html [1] Gardner TS, Cantor CR and Collins JJ] "Construction of a genetic toggle switch in Escherichia coli", Nature 403:339–342, 2000