Team:NCTU Formosa/CI design

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High Temperature Induced System – cI Promoter & cI repressor

Design

In order to make our target protein has a large amount of performance at high temperature, we use the circuit BBa_K098995, which is designed by Team Harvard 2008. (Figure1)

Figure 1.
Part BBa_K098995 Design, with constitutive strong promoter BBa_J23114 , followed by RBS(Ribosome Binding Site) BBa_B0034, cI repressor BBa_K098997, terminator BBa_B0014, and high temperature-sensitive cI promoter BBa_R0051. Among this circuit, cI repressor(BBa_K098997) will be produced constantly to inhibit the cI promoter(BBa_R0051) at low temperature. At high temperature, cI repressor(BBa_K098997) is still produced, however; cI repressor(BBa_K098997) will degrade soon. In this way, cI promoter(BBa_R0051) will be activated. This part is designed by 2008 Harvard on backbone pSB1A2, and we transform this part to host strain DH5α for further use.

This circuit uses gene BBa_K098997coding for cI repressor to inhibit the cI promoter BBa_R0051. The activity of cI repressor is gradually decreased by elevating temperature from 30 ℃ to 42 ℃. We just need to place our target gene after this circuit BBa_K098995, and then regulate the performance of our target protein by incubating E.coli at different temperatures between 30℃ to 42℃. At higher temperature, our target protein will produce abundantly. On the contrary, at lower temperature, it will not express well. The function of this circuit is illustrated as followed. (Figure 2.) At lower temperature, cI dimer (cI repressor, BBa_K098997) will block the RNA polymerase from further transcription, which leads to the gene after this circuit BBa_K098995 can’t be expressed. Although, at higher temperature, cI dimer (cI repressor, BBa_K098997) will degrade and RNA polymerase can transcribe the DNA sequence again, which means that the expression of the gene after this circuit can be recovered.

Figure 2.
First, BBa_K098995 on backbone PSB1A2 is transformed to host strain DH5α. Then we can find out that, at lower temperature, cI dimer (cI repressor, BBa_K098997) will block the RNA polymerase from further transcription, which leads to the gene after this circuit BBa_K098995 can’t be expressed. Although, at higher temperature, cI dimer (cI repressor, BBa_K098997) will degrade and RNA polymerase can transcribe the DNA sequence again, which means that the expression of the gene after this circuit can be recovered.