Part:BBa_K2969013

CI434-TEVsite

An engineered CI transcription factor. We insert the TEV protease recognition sequence between N-terminal and C-terminal of CI434 BBa_C0056. Because the structure of CI434 has been characterized, the function of the fragment between N-terminal and C-terminal is linking. So, we choose this inserting site to avoid bigger influence. In our project, this kind of CI434 can be cleaved by TEV so that it will lose activity.

Characterization

We inserted the TEV protease recognition sequence between N-terminal and C-terminal of CI434 so that it can be cleaved by TEV and then lose activity. We put the reporter gene sf-GFP under the promoter of CI434 so that when TEV protease cleaves CI434, the expression of sf-GFP will rise. We use pTac to regulate the expression of TEV protease. Induced by different concentrations of IPTG, we get the cleavage effciency of TEV protease through the fluorecence of sf-GFP.

Figure 2:The cleavage efficiency of TEV to CI434-TEVsite induced by a series of concentrations of IPTG

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.

Figure 1:the orthogonality among four proteases represented by the fluorescence of sf-gfp is shown.

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
  COMPATIBLE WITH RFC[1000]