Team:UCAS-China/Contribution

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Contribution

We characterized the performance of previous parts, BBa_K2969020 (CI434ts-TEVsite) and BBa_K2969001 (TEVts#6). The newly measured data has been placed on the corresponding part page.

We added more characterization data to the parts we made last year in similar or different ways.

CI434ts-TEVsite is a thermo-sensitive mutation type of CI434 transcription factor.

Contribution of UCAS-China in 2020

We got the quantitative measurement of the cI434 variant and its controls: cI434wt is the wild-type cI434 repressor with an inserted TEV cleavage site; cI434ts is a selected cold-inactivated cI434 repressor. The cold-inactivated cI434 variant (cI434ts) exhibited approximately 3-fold induction when the temperature was shifted from 37 to 25°C.

Fig. 1 The cold-inactivated cI434 variant (cI434ts) exhibited approximately 3-fold induction when the temperature was shifted from 37 to 25°C.

For part BBa_K2969001, we characterized it when we did the experiments about doc 2.0. doc 2.0 is doc 1.0 with a SsrA tag on the C-terminus linked with TEV site. With our cold-inducible on-switch, replacing doc 2.0 with doc 1.0 will make the escape rate lower. The experimental results are in line with the expected hypothesis, indicating that TEV protease does play a role in this process.

Fig. 2 The construction and characterization of cold-inducible kill switch. (a) Genetic circuit design of cold-inducible kill switch (doc 1.0). The protease tevts is cloned under pR to a p15A plasmid. The transcription factor is cloned under a strong promoter J23119 with toxin doc cloned under pR downstream to a pSC101 plasmid. (b) The escape rate of TOP10 doc 1.0 and TOP10 control at 25℃. (c) Genetic circuit design of advanced cold-inducible kill switch (doc 2.0). A hybrid tevS/ssrA tag is fused to the C-terminal domain of Doc toxin. (d) The escape rate of TOP10/EcN doc 1.0 and TOP10/EcN control at 25℃ and 30℃.