Part:BBa_K2969001

TEVts6#

TEVts6# is a thermo-sensitive mutation type of TEV protease. It is created through an evolutionary strategy which use temperature as a screening condition. ‘ts’ means thermo-sensitive while 6# is the number to \rom other mutation types. When the temperature is between 30℃ and 37℃, the protease will gradually lose activity. When it is above 37℃, the activity will become less than the 1/100 of the activity below 30℃.

Characterization

TEVts mutants played an improtant role on switches of UCAS-China 2019 project which is a part of our cold-inducible ON-switches.

We firstly used ELIASA to characterized the tendency of fluorescence change of a series of cold-inducible ON-switches using different TEVts mutants under different temperatures. As shown in Figure 1, all five switches show high fluorescence under low temperature, while their fluorescence all decreases when temperature rises. The groups of TEVts#11, TEVts#17 and TEVts#18 began to inhibit the expression of fluorescence at 37℃ and nearly inhibit the expression of fluorescence completely at 42℃. While the groups of TEVts#6 and TEVts#7 begin inhibition at 30℃ and achieve complete inhibition at 37℃.

Figure 1:The tendency of fluorescence change of a series of TEVts mutants under different temperatures (measured by ELIASA)

Then we chose two kinds of cold-inducible ON-switches, consisting of TEVts#6 and TEVts#18 correspondingly, to measure their temperature response curve more precisely by flow cytometer in TOP10 strain. From Figure 4 we can see that two group both show narrow transition ranges and have ∼100-fold induction, which was achieved within less than ten degrees. Thus, our cold-inducible ON-switches show high performance and versatility, which ensures the potential for basic research, as well as industrial and biomedical applications, and truly makes engineered bacteria precisely controlled.

Figure 2:The induction curve of the cold-inducible ON-switches (TOP10)

Considering our platform is going to serve microbial therapeutics, we found a more suitable E.coil strain, Nissle 1917, a probiotic with more than 100 years of medical application. We also measured the best one, consisting of TEVts#18, in Nissle 1917. It also show high performance similar with it in TOP10 strain.

Figure 3:The induction curve of the cold-inducible ON-switches (Nissle 1917)

Reference

Zheng, Y., Meng, F., Zhu, Z., Wei, W., Sun, Z., Chen, J., . . . Chen, G.-Q. (2019). A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic Acids Research. doi:10.1093/nar/gkz785

Contribution by UCAS-China 2020

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.

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Fig. 1 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℃.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 319
  Illegal SapI.rc site found at 667