Team:DTU-Denmark/Project
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BackgroundWhat 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:
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. IntroductionAimThe 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 ConceptAs 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. 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. ApplicationsIf 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.
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 switchWe 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:
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] 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):
For an in depth description of the function and origin of the regulatory parts see the switch section. Characterizing phage regulatory mechanismsDue 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 2. 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. ConclusionWe 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
- (Jensen 2004) Ole Nørregaard Jensen, “Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry,” Current Opinion in Chemical Biology 8, no. 1 (February 2004): 33-41.
- [1] http://syntheticbiology.org/FAQ.html
- [2]http://www.nature.com.globalproxy.cvt.dk/nrg/journal/v6/n7/execsumm/nrg1637.html
- [3]http://www.nature.com.globalproxy.cvt.dk/msb/journal/v2/n1/full/msb4100073.html
- [4]www.partsregistry.org