Team:DTU-Denmark/Project

From 2010.igem.org

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<p align="justify">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):</p>
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<p align="justify">The switch 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 Gifsy phages were used to circumvent the use of UV-light.</p>
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<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):
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<li>The first level is negative feed back control, repressing the other side of the switch.</li>
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<li>The first level is negative feed back control - repression of the uninduced state of the switch.</li>
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<li>The second mechanism is a positive feed back mechanism that has a threshold level that when triggered will induce the third level of regulation.</li>
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<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>
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<li>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.</li>
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<li>The third regulatory level is a positive feed back mechanism stabilizing the expression of the winning state by, anti-repression of the repression in first level.</li>
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</ol>See figure 3</p>
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  <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>
  <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>

Revision as of 11:45, 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 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 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 novel biological switch.

Our switch design is a complex regulatory system, which is induced by different carbon sources. 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.

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 implie 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 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 Gifsy phages were used to circumvent 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 - 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 in first level.
See figure 3

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