Team:DTU-Denmark/Project

From 2010.igem.org

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<li><a href="http://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li><br>
<li><a href="http://2010.igem.org/Team:DTU-Denmark/Switch">The Switch</a></li><br>
<li><a href="http://2010.igem.org/Team:DTU-Denmark/SPL">Synthetic Promoter Library</a></li><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/BBrick_Characterisation">Results</a></li><br>
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<li ><a href="http://2010.igem.org/Team:DTU-Denmark/Modelling">Modelling</a></li><br>
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Revision as of 08:42, 24 October 2010

Welcome to the DTU iGEM wiki!

Background

What is Synthetic Biology?

Not many people have heard of Synthetic Biology. Synthetic biology essentially aims to utilize natures tricks to design and build artificial biological systems for engineering purposes as well as a way to get a better understanding of why biological systems are set up as they are. The term “synthetic biology” was first used on genetically engineered bacteria that were created with recombinant DNA technology. Parts from natural biological systems are taken, characterized and simplified and used as a component of a highly unnatural, engineered, biological system. The term was then used when referring to when organic synthesis is used to generate artificial molecules that mimic natural molecules such as enzymes.

There are two types of synthetic biologists:
  • those who deal with re-designing and fabricating existing biological systems.
  • those who deal with designing and fabricating biological components that do not already exist in the real world.

Synthetic biology provides us with a new perspective from which we can understand and ultimately utilize life for our own benefits. [1,2,3]

For the second type of synthetic biologists, the foundation is to create and characterize ready made biological building parts that can be used in new applications. From the existing list of available biobricks from the partsregistry, it can be seen that currently these functionalities are limited to linear sensor reporter systems, as is the case for inverters, quorum sensing systems and traditional receptor/reporter systems[4]. On This Wiki we present our contribution to the development and characterization of standard biological parts, developed around the frame work of the iGEM competition.



Introduction

Aim

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 two different inputs. Each of the states will have a specific input 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.

Project Concept

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 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.

Applications

If successfully engineered this new technological tool could advance methods used with in many different fields of biological science as environmental engineering, food applications and medico technology, a few suggestions are listed below. Further description of applications see the Switch section under applications.

  • In manufacturing it could be applied as a cheap proof reading or control mechanism in production plants, if the product is exposed to the right or wrong treatment, or an un wanted biological infection or compound, it will change color.
  • In manufacturing or environmental technology it could be used for error tracking or leakage tracking in process plants or in the environment. If the compound to be tracked cannot be measured, or measurements are difficult to obtain, if a biological sensor exists it is possible to track the organism or the reporter downstream of the inducer signal.
  • In food control it could be used as a small piece of tape, containing the modified organism, on the package. It will then be possible to track if the product have been exposed to high temperatures, or unwanted compounds and the consumer can see it directly on the package.
  • A medical application could be to ingest the modified organism. It can then be tested if the organism experience a certain drug, or condition on the way through the intestinal track.
  • In molecular or medical science it could be used for investigations on chemotaxis and biofilm formation experiments, by exposing the biological matrix with, for an example, different light sources, there will be developed layers in the matrix and movements and development can be tracked, in relation to the two different exposures and the time between them.

Our original project idea was developed around the last example, where light (red light at 660nm and blue light at 470nm) would be used as input to induce switching between the two stable states. The initial ideas behind this was to use the bacteria to create artistic drawing or to track movement in biofilm.

Based on this idea, as well as research of biological switches and the regulatory mechanisms found in phages, we discovered 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 [LINK TO MODELING]

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 the experimental setup click here and results from the characterization experiments see under the respective sections in the wiki. [LINKS]

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.

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