Team:BHSF ND/Improve
2019 IGEM BHSF_NDImprovement
Overview
To make use of Bxb1 coding sequence from the parts collection of 2017 Peking iGEM in our recombinase flipping module, we measured the efficiency of such a recombinase system. For the project of 2017 Peking, they employed the recombinase to “turn” the state of the “clock”, “carpiod”, and “controller”, but their sequence was not particularly suited to our project. We aimed to achieve “no leakage” in our system. Therefore, as for the recombinase module, a more absolute turn is needed. We turned our sight to the RBS ahead the coding sequence of Bxb1, and we tried to decrease the strength of the RBS by two commonly used methods: 1.The first method we used is to calculated strengths of different RBS using RBS calculator and built up an RBS library to gain the weakest RBS strength and thereby reduced the expression leakage(In addressing to our project)of Bxb1; 2. The second method is that we mutated the original RBS sequence, screened them and changed the start codon of RBS from ATG to GTG in order to reduce recombinase expression leakage levels(In addressing to our project). We chose the optimal one and retested them. After the improvement we retested the effectiveness of the system again quantitatively by flow cytometry. Qualitative data and quantitative data are provided in the part registry page.