Difference between revisions of "Part:BBa K2969010"
 
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An engineered CI transcription factor. We insert the TVMV protease recognition sequence between N-terminal and C-terminal of CI. Because the structure of CI 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 CI can be cleaved by TVMV so that it will lose activity.
 
An engineered CI transcription factor. We insert the TVMV protease recognition sequence between N-terminal and C-terminal of CI. Because the structure of CI 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 CI can be cleaved by TVMV so that it will lose activity.
  
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<h2>Characterization
 
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<p> 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).
 
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<p>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 CI to build the relation between protease and transcription factor. Then we transform all the 16 kinds of combination of protease and CI into E.coli using sf-gfp as the reporter gene (GOI). Only the right pair can erase the inhibition of CI 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.
 
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<div>[[File:T--UCAS-China--zlc6.png|700px|thumb|center|<b>Figure 1:</b>the orthogonality among four proteases represented by the fluorescence of sf-gfp is shown.]]</div>
 
  
  

Latest revision as of 17:47, 19 October 2019


CI-TVMVsite

An engineered CI transcription factor. We insert the TVMV protease recognition sequence between N-terminal and C-terminal of CI. Because the structure of CI 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 CI can be cleaved by TVMV so that it will lose activity.


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]