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Team:NCTU Formosa/project
From 2013.igem.org
A multiple regulated-system was built using three different regulation mechanisms including red light, temperature, and sRNA. In other words, it is multitasking genetic engineered machine that can express a variable genes depending on the different command given.
Contents |
Introduction
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sRNA
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37 degree Celsius RBS
Unlike normal RBS, 37oC RBS has a unique hairpin secondary structure that encloses the Shine Dalgarno site (SD site), preventing ribosome starting translation. Such structure is favored as it lowers the free energy of the RBS. And since the structure is sustained by base pairing, heat can be employed to break the hydrogen bonds. With the bonds broken, the hairpin would be unfolded, causing the SD site to be exposed and available for ribosome binding. This means that by raising temperature to a certain threshold, 37oC RBS can function as a normal RBS. The threshold of 37oC RBS employed, K115002, is 37oC, implying that K115002 would remain as hairpin structure under 37oC and only functions normally above or at 37oC. This way, the expression of the genes downstream of 37oC RBS can be regulated by temperature.
Light regulator
Light Control
Our system is consisted of two independent parts: light induced part and dark induced part. Each part is capable of expression two different genes depending on the conditions in which the bacteria are grown; and therefore, a total of four different gene expression can be regulated.
Light induced part
At 30°C , Pred is activated by red light and translation proceeds. However, ribosomes can only bind to the normal RBS, as 37°C RBS forms a hairpin structure, forestalling ribosomes from binding. The only gene downstream of the normal RBS is RFP, so only RFP would be expressed. On the other hand, 37°C RBS unfolds at 37°C , resulting in the expression of both LuxR and GFP. LuxR would binds with AHL to form a complex that activates Plux. The activation of Plux produces sRNA that binds to the normal RBS, blocking it from ribosomes. As a result, RFP would not be expressed and only GFP remains at 37°C .
Without red light, Pred would not be activated, and therefore, this system is completely shut down in the dark.
Dark induced part
Pred activates in the presence of red light, producing tetR that represses Ptet. In other words, the dark induced part is inactive in the exposure of red light and active without red light. At 30°C in the dark, only normal RBS functions to express YFP while 37°C RBS remains as a hairpin structure. At 37°C, however, 37°C RBS unfolds to express LuxR and BFP. Just like the mechanism employed in the light induced part, LuxR forms a complex with AHL to activate Plux that produces the sRNA to block normal RBS. This way, the YFP downstream of YFP cannot be expressed and BFP is expressed at 37°C
Future Work
The system we have created in this project is consisted of two different parts: light induced part and dark induced part. Each part regulates two different genes, resulting in a total of four genes that can be regulated. By adding new parts to the system, it is plausible that the system can regulate more genes. We tend to do that by employ more light sensing promoter. Each light sensor we add, we would be able to regulate two more different genes by employing regulation mechanism of 37°C RBS and sRNA.
On the other hand, we tend to optimize our system to achieve subtle control of the four gene expressions. We might be able to do this by taking the values between the limits we have set. Instead of 30°C and 37°C , we tend to take the values between so we can create a lot more conditions under which different level of expression can be achieved. We can also vary the intensity of red light to control the level of expression. The ultimate goal is to precisely control the level of expression of each gene.
