Team:DTU-Denmark/Project

From 2010.igem.org

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<br><li><a href="http://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a></li><br>
<br><li><a href="http://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a></li><br>
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<li ><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling">Modelling</a>
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<li ><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling">Modeling</a>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#Introduction">Introduction</a></li>
<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#Introduction">Introduction</a></li>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#Approach">Modelling Approach</a></li>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#Approach">Modeling Approach</a></li>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#SPL">Modelling SPL</a></li>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#SPL">Modeling SPL</a></li>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#AntiRepressors">Modelling Anti-Repressors</a></li>
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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling#AntiRepressors">Modeling Anti-Repressors</a></li>
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Revision as of 08:46, 25 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 revolved around using light-receptors to instigate the switch between the two stable states. 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 second reporter will be suppressed by repressor 1 which is coexpressed with the first reporter. Conversely, production of the second reporter would be induced by blue light (470 nm).

Figure 1: Simple bistable switch.


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.

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 a colony is killed when the switch is induced by UV-light [5]. 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 [6]. Another general problem with the construction of synthetic switches is the loss of function over time [7]. 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 biological switch.

Design of our switch

We have set up the complete design for a bistable switch. The main design criteria has been that is should remain stable through subsequent generations, which implies that:

  • It should be designed with out induction by UV-light, and 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 two logical NOR-gates typically used when representing electronic circuits. The NOR-gates are integrated within a SR flip-flop switch as illustrated in Figure 1. This switch has two different, mutually exclusive outputs, induced by two different inputs. The last set output will stay on even when the input signal ceases. For further description on the logical behavior and requirements of switches see the modeling section

.

Figure 2: SR flip-flop switch.


The switch is constructed based on phage regulatory mechanisms, that function when inserted into the chromosome (prophages). Furthermore, we used the Gifsy phage repressor - anti-represor system to circumnavigate the use of UV-light. 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, repressing the other side of the switch.
  2. The second mechanism is a positive feed back mechanism that has a threshold level that when triggered will induce the third level of regulation.
  3. The third regulatory level is a positive feed back mechanism increasing the expression of the winning side by, repression of the opposite sites repression, in step one.

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.

Characterizing phage regulatory mechanisms

Due to the complexity of the regulatory circuit design, it was out of the scope of this project to construct the entire switch so focus was put on characterizing the key regulatory subparts needed for successful system function.

The main regulatory parts are the anti-terminator function from lambda phage, and the repressor system from Gifsy phages, see Figure 4 and Figure 5.

Figure 4: Graphical presentation of the anti-terminator part of our regulatory system from the lambda phage.


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


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.

Further we have developed and demonstrated the functionality of a Synthetic Promoter Library, compatible with the BioBrick standard, that can find multiple applications and be used for characterization of BioBricks.

We hope from this work to inspire and give ideas about a possible construction of a genetic switch and hope that it will be possible for next year’s teams to build on our work, benefit from the Synthetic Promoter Standard, investigate missing functionality of our switch and be able to assemble the entire regulatory system.

References

  • (Gottesman et.al. 2002) Gottesman. Max E, Nudler. Evgeny, 2002 ”Transcription termination and anti-termination in E.coli” Genes to cells. (a good introduction review to termination function)
  • (Franklin et.al. 1989) 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