Composite

Part:BBa_K3175001

Designed by: Morris Huang   Group: iGEM19_British_Columbia   (2019-10-12)
Revision as of 03:59, 22 October 2019 by Samuelking (Talk | contribs)


Plasmid vector for SIGEX screening

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.

1) https://www.ncbi.nlm.nih.gov/pubmed/18600226


<img src="T--british_columbia--sample8bar.PNG">

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
    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


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