Difference between revisions of "Part:BBa J100205"
 
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We designed repClone Red to test the function of repressors. Internal BsaI sites allow the insertion of the Ptet promoter and removal of the backwards P2 promoter pointed towards GFP. Successful cloning of the Ptet promoter will change colonies from green to not green. Variations on the Ptet promoter, or introduction of anhydrotetrocycline (aTc), will affect the activity of the repressor, as indicated by the fluorescence of colonies containing the plasmid. This can be used in synthetic biology laboratories or for undergraduate introductory biology lab modules. <br>
 
We designed repClone Red to test the function of repressors. Internal BsaI sites allow the insertion of the Ptet promoter and removal of the backwards P2 promoter pointed towards GFP. Successful cloning of the Ptet promoter will change colonies from green to not green. Variations on the Ptet promoter, or introduction of anhydrotetrocycline (aTc), will affect the activity of the repressor, as indicated by the fluorescence of colonies containing the plasmid. This can be used in synthetic biology laboratories or for undergraduate introductory biology lab modules. <br>
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We found that JM109 spontaneously deleted the transcriptional terminator (TT). This part was more stable, the cells grew better and mini preps yielded more plasmid. Therefore we recommend people use [https://parts.igem.org/Part:BBa_J100324 J100324] instead.  
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We found that JM109 can spontaneously deleted the transcriptional terminator (TT). This part was more stable, the cells grew better and mini preps yielded more plasmid. Therefore people might want to use [https://parts.igem.org/Part:BBa_J100324 J100324] instead. The caveat is that without the TT, the portion removed by BsaI digestion is 60 bp and therefore would be the same size as a new Ptet promoter. See [http://gcat.davidson.edu/GCATwiki/index.php/PCR_for_Bio113 the Bio113 web page for implications for PCR screening of new promoter cloning]. 
  
 
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Latest revision as of 19:41, 2 August 2017

repClone Red

We designed repClone Red to test the function of repressors. Internal BsaI sites allow the insertion of the Ptet promoter and removal of the backwards P2 promoter pointed towards GFP. Successful cloning of the Ptet promoter will change colonies from green to not green. Variations on the Ptet promoter, or introduction of anhydrotetrocycline (aTc), will affect the activity of the repressor, as indicated by the fluorescence of colonies containing the plasmid. This can be used in synthetic biology laboratories or for undergraduate introductory biology lab modules.
We found that JM109 can spontaneously deleted the transcriptional terminator (TT). This part was more stable, the cells grew better and mini preps yielded more plasmid. Therefore people might want to use J100324 instead. The caveat is that without the TT, the portion removed by BsaI digestion is 60 bp and therefore would be the same size as a new Ptet promoter. See [http://gcat.davidson.edu/GCATwiki/index.php/PCR_for_Bio113 the Bio113 web page for implications for PCR screening of new promoter cloning].

J100205.png

Variations from the registry sequence for TetR and GFP were sequence verified, and functionality was experimentally verified.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 9
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 9
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 9
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 9
    Illegal AgeI site found at 2288
    Illegal AgeI site found at 2400
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1638
    Illegal BsaI.rc site found at 1517