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We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37° C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy.

First, we use the constitutive promoter, 37° C induced RBS (K115002), the tetR gene and terminators. We will call this part the “Regulator”.

We use the “Regulator” to maintain a stable growth rate of bacteria while inhibiting crystal protein protection by way of the tetR gene. Bacteria growth, particularly vector mutation rate and growth rate, is highly dependent on amplification conditions, thus crystal protein production is off during the incubation/amplification step. Because the tetR gene of the “Regulator” can block the pTet promoter of “Production”, we achieve a self-regulating, temperature dependent system that monitors both bacteria growth and protein production.

We speculate that when the “Regulator” and “Production” sections are combined, we can achieve a high bacteria growth rate and close to no crystal protein producing rate at T>37° C, and low bacteria growth rate and high crystal protein producing rate at T<37° C.