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Team:NCTU Formosa/Design
From 2010.igem.org
Project>Design
Design Overview
Fig. 1. The circuit design of Mosquito Intelligent Terminator. The Cry weapon system is located on strand B. The Low temperature release system is controlled by strand A and B. The population control system is controlled by strand C and D
The main concept of the Mosquito Intelligent Terminator (MIT or Terminator) is to insert cry gene isolated from Bacillus thuringiensis subsp. Israelensis into the E.coli. E.coli with this gene can produce Cry proteins toxic to mosquito larva. When the temperature exceeeds 37°C, the temperature-sensitive RBS BBa_K115002 is activated and produce tetR proteins inhibiting the Ptet promoter. This mechanism serves to regulate the cry protein release in a temperature dependent fashion, where the Ptet promoter is inhibited. On the flip side, when the temperature is below 37°C, the cry proteins will be produced. The whole reaction can be divided into the following steps:1. Reproduction status (>37°C): The E.coli population grows and produces tetR proteins to inhibit the Ptet promoter.
2. Downstream protein release status (<37°C): E.coli released into environment tetR begins to degrade, Ptet promoter actively producing cry proteins, luxI & luxR.
3. The self-maintain status ( < 37°C): When external bacteria aggregate, the concentration of AHL will rise pass threshold level and resulting the formation of AHL-LuxR complexes. This complex then becomes a translation factor to Plux promoter and expression of downstream ccdB gene is activated. Accumulation of ccdB protein result in subsequent suicide. Therefore, when the population size of E. coli rises pass the threshold level, ccdB proteins are induced by AHL to restrict the population size.
A,B Device
A (Low-temperature control system)
J23101+RBS(BBa_k115002)+tetR+terminator
The theoretical switching temperature of RBS BBa_k115002 was designed by Team TUDelft in 2008 is37°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 (Cry weapon system)
pTet+RBS+cry+RBS+GFP+terminator
The "crystal protein producing device", B, works below37°C and produces the crystal protein. It can be repressed by the aforementioned thermosensing device A. Low-temperature control system A. In order to test the function of A and pTet, we constructed B with GFP (green fluorescence protein) gene behinds cry gene. With the expression of green fluorescence protein, the function of A and promoter pTet can be verified.
C,D Device
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.
