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A (Thermosensing Device)

J23101+RBS(k115002)+tetR+terminator

The theoretical switching temperature of RBS BBa_k115002 was designed by Team TUDelft in 2008 is 37 C. Thus, our thermosensing device A can be induced at this temperature. (The switching temperature can be modified by changing RBS according to different conditions.) While being cultured above 37 C, BBa_k115002 is supposed to be induced. Once swiched on, tetR will inhibit the corresponding promoter, pTet, which enables us to regulate the expression of cry gene and quorum sensing gene devices. In the mosquito-infested areas below 37 C, device A will not work after spreading, resulting in the production of cry proteins, AHL, and LuxR proteins.

B (Crystal Protein Producing Device)

pTet+RBS+cry+terminator

The "crystal protein producing device", B, works below 37 C and produces the crystal protein. It can be repressed by the aforementioned thermosensing device A.

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Another consideration was the effect of the cry protein on E. coli production. Accumulation of these proteins may affect their growth, therefore, we use the tetR gene, followed with a 37。C induced RBS, as inhibitor for the cry gene expression. At the incubation temperature of 37。C, the RBS activates and tetR inhibit Ptet, which then inhibits the expression of cry gene. Only after we spread the E. coli to wriggler infested areas, temperatures drop below 37。C and Ptet inhibition is released, resulting in cry protein production. The next consideration was how to effectively control our E. coli population. To address population control, we introduced the LuxI/R system from V. fischeri, specifically genes ccdB, luxR and luxI. SAM is one of the metabolic products of E. coli. LuxI is a synthase that converts S-adenosylmethionine (SAM) into a small molecule called an acyl-homoserine lactone (AHL) and LuxR is a constitutively expressed protein that can bind AHL, which form a complex for activating Plux. ccdB are produced after Plux is activated and when in combination cause E. coli self-destruction. When the concentration of AHL and luxR rises, the Plux has a higher activation rate, resulting in ccdB production and subsequent suicide. This population control mechanism is not activted during E. coli incubation, because luxR and luxI are also initiated by Ptet. ptet is inhibited by the temperature sensitive RBS and tetR, and during incubation is off by default. In our initial experiment, we use GFP and mRFP to substitute for cry and ccdB respectively. This makes it our design easy to test and observe.

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