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Revision as of 20:14, 18 September 2015

　

Biosafety

Light-induced lysis system
ExTermite Coli targets to inhibit the metabolic pathway of termite. However, most of insects store their energy in form of trehalose, the same as termite. Therefore, there is one possibility that ExTermite Coli affect other insects. Also, we have to deal with the problem of E. coli spread into the environment. As we use pseudo egg to carry ExTermite Coli to termite’s colony, there is a possibility of distribution and that may cause a genetic pollution.
To avoid these problems, we add another function to ExTermite Coli….light-induced lysis system.
　　　　　
The reason for constructing “light-induced” lysis system.
One of the most characteristic features of termite is negative phototaxis. When termites such as Coptotermes formosanus exposed to the light, they move very slowly or they stop to move. As for Hodotermopsis sjostedti, they started to run away from the light. Because of termite’s negative phototaxis, ExTermite Coli should be designed to work under dark place.
In other words, ExTermite Coli shouldn’t work under light place to avoid damaging other insects outside of house.
Therefore, to prevent the risk of damaging other insects, we constructed light-induced lysis system.
Mechanism of light-induced lysis system
　To construct our biosafety system, we choose two components.
　First, we choose cpcG2 promoter to induce gene expression by light. CpcG2 promoter originates from cyanobacteria, which can response to green light. In cyanobacteria, cpcG2 promoter function together with other two response regulators, CcaR and CcaS. By introducing this system into E.coli that does not have any photoreceptor, we can induce expression of a specific gene in E.coli.
　　　
　Second, we select the Colicin-E1 as a lysis protein. Colicin-E1 is a membrane protein originating from E.coli. Colicin-E1 forms multimer and binds to receptor on outer membrane to create a pore. It causes membrane disruption and cell lysis.
　 Using these two components, we constructed light-induced lysis system.
Construction of light-induced lysis gene and evaluation
　 First of all, we constructed following Biobricks containing Colicin-E1(BBa_K1765002).
We constructed our biobrick from BBa_K822002, which is coding Colicin-E1 and its immune protein. After cloning the genes of Colicin-E1 from this biobrick, we attached T0 terminator and cpcG2 promoter (BBa_K1765002).
　After transformation of these Biobricks into E. coli, we monitored growth of the transformants.