Difference between revisions of "Part:BBa K3175001:Design"

 
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===Design Notes===
 
===Design Notes===
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The vector-trap plasmid was designed to ensure the maximum possibility of acquiring a gene that may contain the promoter-transcription factor combination that we want. We were inspired by Uchiyama and Watanabe’s plasmid vector design, with multiple changes(1). By using a blunt end restriction site (NruI), we ensured that a higher variety of DNA would be ligated. We compensated for the lower ligation efficiency through fast-link DNA ligase and blunt-end fixing for the prepped metagenomic DNA. As per the paper, a GFP gene was added for possible FACs screening, but due to blunt ends ligate with different orientations, a reverse RFP was added as well. The reporter genes allowed FACs to happen for high-throughput screening of the fluorescing cells if a promoter were to be ligated into the vector.
  
 
===Source===
 
===Source===

Revision as of 08:33, 21 October 2019


Plasmid vector for SIGEX screening


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 7
    Illegal AgeI site found at 119
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1382


Design Notes

The vector-trap plasmid was designed to ensure the maximum possibility of acquiring a gene that may contain the promoter-transcription factor combination that we want. We were inspired by Uchiyama and Watanabe’s plasmid vector design, with multiple changes(1). By using a blunt end restriction site (NruI), we ensured that a higher variety of DNA would be ligated. We compensated for the lower ligation efficiency through fast-link DNA ligase and blunt-end fixing for the prepped metagenomic DNA. As per the paper, a GFP gene was added for possible FACs screening, but due to blunt ends ligate with different orientations, a reverse RFP was added as well. The reporter genes allowed FACs to happen for high-throughput screening of the fluorescing cells if a promoter were to be ligated into the vector.

Source

filler

References