Improvements
Parts Improvement: Solve the expression burden and high leakage expression problem by constructing and appling mutant inducible promoters
In experiment, we found that the expression of several sorts of recombinases will undermine the growth of E.coli and reduce the efficiency and accuracy. In the graph 1 below, determination on the concentration of E.coli shows that as the concentration of Int8 and TG1 increase, the growth of E.coli has been significantly restrained. For Int8, according to the data, when the concentration of Int8 surpass 30000, it will influence the growth of cell. Hence, we have to improve this promotor and ensure that at its highest output to recombinase, it won’t produce toxicity. When it’s producing lowest output, the expression of recombinase is still able to recombinase the DNA. So, we mutated the Ptac promotor and get the data(Figure 2):
Figure 1
Figure 2
Figure 3
Ptac M1, M2, M3 are three mutation of Ptac and each has different output
intensity. We choose Ptac M2, the promotor which output is neither too high
nor too low. Then, we combine it with K3254011 and test its toxicity to the
cell. We found that after mutation, the toxicity profoundly
decreases（figure 3）.
Project Improvement: Extend the function of integrase based genetic switch
Our project can be seen as an improvement for part of the Peking's iGEM project in 2017. Based on that project, we achieved:
1,Extending the function of att sites-terminator based genetic switch. Two BsaI restriction sites were added between attB site and
terminator. As a result, it can work as a normally closed (NC) switch for
the gene which was inserted between the two BsaI sites and switch to OFF
state when it flipped or excised.
2, In addition to treating the recombinase system as a simple response at two input levels, our project also achieved the modification and utilization of the response interval of the system.
3, Extending the well characterized recombinase library.
