Team:DTU-Denmark/Project

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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>
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<p align="justify">For an in depth description of the function and origin of the regulatory parts have a look into the <a href=" http://2010.igem.org/Team:DTU-Denmark/Switch" target="_blank">switch</a> section.</p>
<p align="justify">For an in depth description of the function and origin of the regulatory parts have a look into the <a href=" http://2010.igem.org/Team:DTU-Denmark/Switch" target="_blank">switch</a> section.</p>

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