Team:DTU-Denmark/Project

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<title>Welcome to the DTU iGEM wiki!</title>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark" >Home</a></font> </td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark" >Home</a></font></td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Team" >The Team</a> </font></td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Team" >The Team</a> </font></td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Project" >The Project</a> </font></td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Project" >The Project</a>  
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Parts" >Parts submitted</a> </font></td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Parts" >Parts submitted</a> </font></td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Modelling">Modelling</a></font> </td>
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     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Results">Results</a></font> </td>
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Notebook" title="Day to day lab activity">Notebook</a>  
     <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Notebook" title="Day to day lab activity">Notebook</a>  
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  <td align="center" ><font face="arial" size="3"><a class="mainLinks" href="https://2010.igem.org/Team:DTU-Denmark/Blog">Blog</a></font> </td>
</font></td>
</font></td>
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  <font color="#990000" face="arial" size="3">
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<ul type="circle">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Background">Synthetic Biology</a></li><br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems">Regulatory Systems</a></li>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#lambda">Lambda Phage</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems#gifsy">Gifsy Phages</a></li>
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</font></ul>
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<br>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Biological_Switch">Biological Switches</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Bistable_Switches">Bistable Switches</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Design">Design of our Bi[o]stable Switch</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Engineering">Step-wise Engineering of the Switch</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Switch#Applications">Applications</a></li>
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</font></ul>
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<br><li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#standard">The DTU SPL Standard</a>
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<ul>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#strategy">Strategy</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#design">Primer Design</a></li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#protocol">Protocol</a></li>
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</ul>
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</li>
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/SPL#advantages">Advantages</a></li>
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</ul></font>
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</li><br>
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<li ><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling">Modeling</a>
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<ul><font size="2">
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<li><a href="https://2010.igem.org/Team:DTU-Denmark/Modelling#Approach">Modeling Approach</a></li>
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</ul></font>
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</li><br>
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</ul>
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  <td width="556px" height="100%" valign="top">
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  <font color="#990000" face="arial" size="5">
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<br> 
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<b>Project Concept</b><br><br>
 +
  </font>
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<p align="justify">As previously stated, the main goal of our project is to design a bistable switch. We want to enable bacteria to transition between two stable states. In our system, switching between states will be induced by two different inputs and each of the states will have a specific output associated with it.</p>
 +
 +
<p align="justify">Our original project concept evolved around building a switch that we could turn on and off continuously. Not only did we want the switch to be able to switch states, but we also wanted it to be able to stay in a certain state without having to induce it constantly. Several designs were discussed, for example using light at different wavelengths to induce the system.</p>
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<p align="justify">During the last couple of years several attempts have been made to construct bistable switches. One switch design is a one input, two outputs stable switch. It has a stable output but it looses the switching ability and 90% of the individuals in the population are killed when the switch is induced by UV-light (Lou, C. et al.,2010). Another mechanism tested has been a flipases system where the DNA is inverted by specific recognition sites. The system was found to function but was limited by the robustness of the flipase systems and knowledge about their function (Ham, T.S. et al.,2008). Another general problem with the construction of synthetic switches is the loss of function over time (Canton, B. et al.,2008). The limited function and stability of existing switches also limit the application to short time spans. Based on these problems we saw the untapped potential in designing a novel biological switch.</p>
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</body>
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<p align="justify">Our switch design is a complex regulatory system, which is induced with the help of input plasmids carrying inducible promoters.  However due to the complexity of the design of the bistable switch it was out of the scope of this project to construct the entire switch. Therefore focus was put on characterizing the key regulatory subparts needed for successful switch function. Characterizing subparts also enable future teams to use them in other contexts.</p>
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</html>
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<table class="https://static.igem.org/mediawiki/2010/9/9a/Intro_switch.png" align="center">
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<caption align="bottom"><p align="justify"><b>Figure 1</b>: A simplified illustration of our bistable switch.</p></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2010/9/9a/Intro_switch.png"  width="400px"></td></tr>
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= '''Bacterial''' ''Light'' '''Switch!''' =
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<font color="#990000" face="arial" size="5">
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<br> 
 +
<b>Design of our switch</b><br><br>
 +
  </font>
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== '''Introduction''' ==
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<p align="justify"> We have set up the complete design for a bistable switch. The main design criteria has been that the switch should be able to toggle  back and forth between states, stay in its induced state until it receives another input and remain stable through subsequent generations. These criteria imply that:
 +
<ul>
 +
<li>It should be designed without induction by UV-light.</li>
 +
<li>It should not be based on essential native regulatory mechanisms.</li>
 +
<li>It should be possible to incorporate into the genome for stable replication, and function in subsequent generations.</li>
 +
</ul>
 +
<p align="justify">A simplified version of our switch design can be illustrated using a basic  SR (Set-Reset) flip flop circuit used when representing electronic circuits. It provides feedback from its outputs to its inputs and is commonly used in memory circuits to store data bits. The term flip-flop relates to the actual operation of the device, as it can be "Flipped" into one logic state or "Flopped" back into another <a href="http://www.electronics-tutorials.ws/sequential/seq_1.html" target="_blank"> (reference)</a>. For further description on the logical behavior and requirements of switches see the <a href="https://2010.igem.org/Team:DTU-Denmark/Modelling" target="_blank">modeling section</a>.</p>
 +
<table class="https://static.igem.org/mediawiki/2010/5/53/SRflipflop.png" align="center">
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<caption align="bottom"><p align="justify"><b>Figure 2</b>: SR flip-flop switch.</p></caption>
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<tr><td><img src="https://static.igem.org/mediawiki/2010/5/53/SRflipflop.png"  width="200px"></td></tr>
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</table><br>
 +
<p align="justify">The switch design is based on phage regulatory systems. We used the repressor/anti-represor system from the Gifsy phages and an anti-termination system from the lambda-phage.</p>
 +
<p align="justify"> The switch has three levels of regulatory mechanisms to ensure a stable expression and tight control and thereby creating a robust bistable switch (see Figure 3):
 +
<ol>
 +
<li>The first level is negative feed back control - repression of the uninduced state of the switch.</li>
 +
<li>The second level is a positive feed back mechanism with a threshold level that when triggered will induce the third level of regulation - antitermination allows third level to be induced.</li>
 +
<li>The third regulatory level is a positive feed back mechanism stabilizing the expression of the winning state by, anti-repression of the repression from the loosing states.</li>
 +
</ol></p>
-
==== '''Overall Goal''' ====
+
<table class="https://static.igem.org/mediawiki/2010/1/14/Threestages.png" align="center">
-
The goal of our project is to enable colonies of ''E. coli'' bacteria to transition between production of two different reporter proteins. In our system, switching between states will be induced by exposing the bacteria to light. Each of the states will have a specific frequency associated with it. There are multiple potential applications for biologicals "switches" such as these, this includes the improved control of production of additives in industrial biotechnological processes.
+
<caption align="bottom"><p align="justify"><b>Figure 3</b>: Simplified representation of the regulatory mechanisms: [<b>1</b>] negative feed back control of opposite side. [<b>2</b>] positive feed back trigger mechanism for side commitment. [<b>3</b>] positive feed back mechanism, by canceling the opposite sides repressor.</p></caption>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2010/1/14/Threestages.png"  width="400px"></td></tr>
 +
</table><br>
 +
<p align="justify">For an in depth description of the function and origin of the regulatory parts have a look into the <a href=" https://2010.igem.org/Team:DTU-Denmark/Switch" target="_blank">switch</a> section.</p>
 +
<p align="justify">One important feature of the switch is the strength of the promoters. For the switch to work properly we need promoters of equal strength. To solve this problem we utilized a <a href=" https://2010.igem.org/Team:DTU-Denmark/SPL" target="_blank">synthetic promoter library</a>, enabling us to generate a library of promoters with a wide variety of different strengths.</p>
-
==== '''Project Concept''' ====
+
<font color="#990000" face="arial" size="5">
-
As previously stated, the main goal of our project is to design a bistable switch. The switching between the two states will be controlled by the introduction of two different wavelengths of light, each wavelength responsible for the induction of a different state. As a proof of concept, we’re using fluorescent proteins as reporter genes which makes it easy to observe and characterise the system. In principle, however, any reporter gene can be used.
+
<br> 
 +
<b>Characterizing phage regulatory mechanisms</b><br><br>
 +
  </font>
-
Our original project concept revolved around using light-receptors to instigate the switch between the two stable state. It was thought that the production of the first reporter protein would be induced by red light (660 nm). At the same time, production of the other reporter will be suppressed by a coexpressed repressor. Conversely, production of the second reporter would be induced by blue light (470 nm). Bistability of the system is achieved by using two repressors which negatively regulate each other’s expression. This enables the system to sustain state without continuous input, i. e. once production of a reporter protein is initiated, it will persist until the system is forced into the other state.
 
-
[[Image:DTU_Project_illustration_1.png‎|400px|center]]
 
-
Our project concept has since changed to using two different carbohydrate sources as a means of switching between the two stable states. This means that the state in which the bacteria will be found depends on which one of two carbohydrate sources it was last exposed.
+
<p align="justify">The main regulatory parts of the switch are the repressor/antirepressor system from the Gifsy phages and the anti-terminator system from lambda phage, see Figure 4, Figure 5 and Figure 6.</p>
-
= '''Background''' =
+
<table class="https://static.igem.org/mediawiki/2010/c/c8/DTU_anitsystem.png" align="center">
 +
<caption align="bottom"><p align="justify"><b>Figure 4</b>: Graphical presentation of the repressor part of our regulatory system from the Gifsy phages.</p></caption>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2010/c/c8/DTU_anitsystem.png"  width="225px"></td></tr>
 +
</table><br>
-
== '''Repressors''' ==
+
<table class="https://static.igem.org/mediawiki/2010/2/26/Repressor_antirepressor.png" align="center">
 +
<caption align="bottom"><p align="justify"><b>Figure 5</b>: Graphical presentation of the anti-repressor part of our regulatory system from the Gifsy phages.</p></caption>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2010/2/26/Repressor_antirepressor.png"  width="350px"></td></tr>
 +
</table><br>
-
==== '''Alpha-repressor''' ====
+
<table class="https://static.igem.org/mediawiki/2010/a/a4/DTU_repressorsystem.png" align="center">
 +
<caption align="bottom"><p align="justify"><b>Figure 6</b>: Graphical presentation of the anti-terminator part of our regulatory system from the lambda phage.</p></caption>
 +
<tr><td><img src="https://static.igem.org/mediawiki/2010/a/a4/DTU_repressorsystem.png"  width="180px"></td></tr>
 +
</table><br>
-
The C1-repressor is responsible for repressing transcription of the lytic genes, thereby maintaining the stable lysogenic state. The induction of the lytic state is caused by activated RecA, which stimulates the self-cleavage of the C1-repressor. We will be using the C1-repressor in our system.
 
-
== '''Transcription Termination and Anti-Termination''' ==
 
-
==== '''Termination''' ====
+
<p align="justify">As a proof of concept for the regulatory mechanisms, we constructed plasmids that were able to test the regulatory mechanism and strength of the two systems. We used low copy number plasmids and fluorescent proteins as reporters. For more information about the experimental setup and characterization results of the Repressor - Anti-Repressor system please click <a href="https://2010.igem.org/Team:DTU-Denmark/Repressor_Section" target="_blank">here</a> and for the Terminator - Anti-Terminator system please click <a href="https://2010.igem.org/Team:DTU-Denmark/AntiTermination_Section" target="_blank">here</a>.</p>
-
Termination can fall into one of two catagories:
+
<p align="justify">The key parts of the regulatory systems have been tested and are available as BioBricks through the parts registry. See the <a href=" https://2010.igem.org/Team:DTU-Denmark/Parts" target="_blank">parts</a> page for a list of available parts. </p>
-
* ''Intrinsic Termination''
+
<font color="#990000" face="arial" size="5">
-
* ''Factor-dependent Termination''
+
<br> 
 +
<b>Conclusion</b><br><br>
 +
  </font>
-
'''Intrinsic Termination''' can be found to occur at defined template sequences, usually a region of hyphenated inverted sequence symmetry followed by a run of T residues. Termination through intrinsic terminators is stimulated by additional factors, e.g. NusA. Termination occurs due to the stem-loop structure formed by the base-pairing of mRNA with itself caused by inverted sequence symmetry, followed by the run of T residues. The NusA protein causes the RNA-p complex to temporarily stall at the stem-loop structure, when this is followed by a poly-A tail, the RNA-DNA duplex is destabilized. This causes the RNA-p to dissociate from the DNA, thereby terminating transcription.
+
<p align="justify">We have shown that the Gifsy repressor system has a sufficient tight expression and control to be used in the future construction of biological switches.</p>
-
'''Factor-dependent Termination''' occurs due to events that are not directly related to transcription, such as the release of ribosomes from nascent transcript or DNA damage. One such host termination factor is Rho, which acts on many sites along the bacterial chromosome. MFD, is a host termination factor that is responsible for releasing RNA-p stalled at sites of UV-induced DNA lesions.
+
<p align="justify">We have set up the frame work for testing anti-terminator function, but further characterization is needed before it can be applied in standard regulatory systems.</p>
-
==== '''Anti-Termination''' ====
+
<p align="justify">Furthermore we have designed and demonstrated the functionality of a Synthetic Promoter Library, compatible with the BioBrick standard. We have also developed a standard for integrating a BioBrick compatible Synthetic Promoter Library in bacteria in order to fine-tune the expression of BioBrick parts and devices.</p>
-
Anti-Termination is the process by which the termination of gene transcription is prevented. Such control of gene transcription can be found in the phage Lambda system. The mechanism is controlled by proteins, such as the lambda N or lambda Q-proteins. The expression of early genes and late genes are both regulated by the anti-termination mechanism, controlled by the lambda N-protein and the lambda Q-protein, respectively.  
+
<p align="justify">We hope that this work will inspire future teams to take up the challenge of constructing a genetic bistable switch. They can easily benefit from the new DTU Synthetic Promoter Library standard and our submitted BioBricks.</p>
-
The N-protein is able to suppress transcription termination at both factor-dependent and factor-independent termination sites. N anti-termination is strongly stimulated by the NusA protein. Unlike the N-protein, the Q-protein specifically binds to a DNA sequence immediately upstream of the pR´ promoter.
+
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A more detailed explanation of these anti-termination mechanisms will be posted later on.
+
<!-- Main content area -->
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+
-
=== N-protein plasmid ===
+
-
The N protein were isolated from salmonella genomic DNA with specific designed primers. We used the natural occurring RBS site, as a High expression of N have shown non specific anti-termination effect on a global scale on the genome. [[#References References]]
+
 +
</body>
 +
</html>
== References ==
== References ==
-
* NC Franklin, JH Doelling - Am Soc Microbiol "Overexpression of N antitermination proteins of bacteriophages lambda, 21, and P22: loss of N protein specificity." - Journal of bacteriology, 1989
+
* Canton, B. et al. Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26, 787-793, (2008)
-
* Ole Nørregaard Jensen, “Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry,” Current Opinion in Chemical Biology 8, no. 1 (February 2004): 33-41.
+
* Ham, T.S. et al. Design and construction of a double inversion recombination Switch for Heritable Sequential Genetic Memory. PloS ONE 3(7),(2008)
 +
* Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol Syst Biol 6,  (2010)
 +
* [1] http://syntheticbiology.org/FAQ.html
 +
* [2]http://www.nature.com.globalproxy.cvt.dk/nrg/journal/v6/n7/execsumm/nrg1637.html
 +
* [3]http://www.nature.com.globalproxy.cvt.dk/msb/journal/v2/n1/full/msb4100073.html
 +
* [4]www.partsregistry.org

Latest revision as of 03:58, 28 October 2010

Welcome to the DTU iGEM wiki!


Project Concept

As previously stated, the main goal of our project is to design a bistable switch. We want to enable bacteria to transition between two stable states. In our system, switching between states will be induced by two different inputs and each of the states will have a specific output associated with it.

Our original project concept evolved around building a switch that we could turn on and off continuously. Not only did we want the switch to be able to switch states, but we also wanted it to be able to stay in a certain state without having to induce it constantly. Several designs were discussed, for example using light at different wavelengths to induce the system.

During the last couple of years several attempts have been made to construct bistable switches. One switch design is a one input, two outputs stable switch. It has a stable output but it looses the switching ability and 90% of the individuals in the population are killed when the switch is induced by UV-light (Lou, C. et al.,2010). Another mechanism tested has been a flipases system where the DNA is inverted by specific recognition sites. The system was found to function but was limited by the robustness of the flipase systems and knowledge about their function (Ham, T.S. et al.,2008). Another general problem with the construction of synthetic switches is the loss of function over time (Canton, B. et al.,2008). The limited function and stability of existing switches also limit the application to short time spans. Based on these problems we saw the untapped potential in designing a novel biological switch.

Our switch design is a complex regulatory system, which is induced with the help of input plasmids carrying inducible promoters. However due to the complexity of the design of the bistable switch it was out of the scope of this project to construct the entire switch. Therefore focus was put on characterizing the key regulatory subparts needed for successful switch function. Characterizing subparts also enable future teams to use them in other contexts.

Figure 1: A simplified illustration of our bistable switch.



Design of our switch

We have set up the complete design for a bistable switch. The main design criteria has been that the switch should be able to toggle back and forth between states, stay in its induced state until it receives another input and remain stable through subsequent generations. These criteria imply that:

  • It should be designed without induction by UV-light.
  • It should not be based on essential native regulatory mechanisms.
  • It should be possible to incorporate into the genome for stable replication, and function in subsequent generations.

A simplified version of our switch design can be illustrated using a basic SR (Set-Reset) flip flop circuit used when representing electronic circuits. It provides feedback from its outputs to its inputs and is commonly used in memory circuits to store data bits. The term flip-flop relates to the actual operation of the device, as it can be "Flipped" into one logic state or "Flopped" back into another (reference). For further description on the logical behavior and requirements of switches see the modeling section.

Figure 2: SR flip-flop switch.


The switch design is based on phage regulatory systems. We used the repressor/anti-represor system from the Gifsy phages and an anti-termination system from the lambda-phage.

The switch has three levels of regulatory mechanisms to ensure a stable expression and tight control and thereby creating a robust bistable switch (see Figure 3):

  1. The first level is negative feed back control - repression of the uninduced state of the switch.
  2. The second level is a positive feed back mechanism with a threshold level that when triggered will induce the third level of regulation - antitermination allows third level to be induced.
  3. The third regulatory level is a positive feed back mechanism stabilizing the expression of the winning state by, anti-repression of the repression from the loosing states.

Figure 3: Simplified representation of the regulatory mechanisms: [1] negative feed back control of opposite side. [2] positive feed back trigger mechanism for side commitment. [3] positive feed back mechanism, by canceling the opposite sides repressor.


For an in depth description of the function and origin of the regulatory parts have a look into the switch section.

One important feature of the switch is the strength of the promoters. For the switch to work properly we need promoters of equal strength. To solve this problem we utilized a synthetic promoter library, enabling us to generate a library of promoters with a wide variety of different strengths.


Characterizing phage regulatory mechanisms

The main regulatory parts of the switch are the repressor/antirepressor system from the Gifsy phages and the anti-terminator system from lambda phage, see Figure 4, Figure 5 and Figure 6.

Figure 4: Graphical presentation of the repressor part of our regulatory system from the Gifsy phages.


Figure 5: Graphical presentation of the anti-repressor part of our regulatory system from the Gifsy phages.


Figure 6: Graphical presentation of the anti-terminator part of our regulatory system from the lambda phage.


As a proof of concept for the regulatory mechanisms, we constructed plasmids that were able to test the regulatory mechanism and strength of the two systems. We used low copy number plasmids and fluorescent proteins as reporters. For more information about the experimental setup and characterization results of the Repressor - Anti-Repressor system please click here and for the Terminator - Anti-Terminator system please click here.

The key parts of the regulatory systems have been tested and are available as BioBricks through the parts registry. See the parts page for a list of available parts.


Conclusion

We have shown that the Gifsy repressor system has a sufficient tight expression and control to be used in the future construction of biological switches.

We have set up the frame work for testing anti-terminator function, but further characterization is needed before it can be applied in standard regulatory systems.

Furthermore we have designed and demonstrated the functionality of a Synthetic Promoter Library, compatible with the BioBrick standard. We have also developed a standard for integrating a BioBrick compatible Synthetic Promoter Library in bacteria in order to fine-tune the expression of BioBrick parts and devices.

We hope that this work will inspire future teams to take up the challenge of constructing a genetic bistable switch. They can easily benefit from the new DTU Synthetic Promoter Library standard and our submitted BioBricks.

References