Part:BBa_K2969002

TEVts7#

TEVts7# is a thermo-sensitive mutation type of TEV protease. It is created through an evolutionary strategy which use temperature as a screening condition. 7# is the number to help differentiate it from 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

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