Introduction

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. Each of the states will have a specific output associated with it.

The switching between the two states will be controlled by the introduction of two different inputs, each input 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.

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.

Figure 1: Simple bistable switch.

Our project concept has since changed to concentrating on the different composite parts of the switch and leave the assembling of the entire switch as an option for next years DTU team.

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 see 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 testes and are available as biobricks through the parts registry. See the parts page for a list of the parts available.

Conclusion

We have shown that the gifsy repressor system have 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.