Part:BBa_K2969006
TVMV protease
TVMV protease is from Tobacco Vein Mottling Virus, which recognizes a linear epitope of the general form T_V_R_F_Q / S with cleavage occurring between Q and S. The protease is used to cleave affinity tag from fusion protein. The optimal temperature for cleavage is 30 °C. In our project we insert the recognition sequence into the transcription factors so that the expression of protease can inhibit the action of transcription factors.
Characterization
In UCAS-China 2019 project, TEV protease is an important part of our switches. According to resent research, we can find many similar proteases to build similar cold-inducible switches such as TVMV protease (cleavage site TVRFQS), SuMMV protease (cleavage site EEIHLQ), HRV3C (cleavage site LEVLFQGP).
To realize the function of sequence switches, we combine the two different cold-inducible switches. However, we should make sure that these two combinations have no influence on each other. Thus, we design experiments to verify the orthogonality of different cold-inducible switch. That is the orthogonality of different proteases and their cleavage sites. We insert all the four cleavage sites into the transcription factor CI434 to build the relation between protease and transcription factor. Then we transform all the 16 kinds of combination of protease and CI434 into E.coli using sf-gfp as the reporter gene (GOI). Only the right pair can erase the inhibition of CI434 and trigger the expression of sf-gfp. Through the results of the fluorescence expression, we can find whether there are interactions between one protease and one cleavage site. The results below demonstrate good orthogonality between these proteases and their cleavage sites.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 366
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 228
- 1000COMPATIBLE WITH RFC[1000]
| None |