Difference between revisions of "Part:BBa K3175001:Design"
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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. | 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. | ||
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===References=== | ===References=== | ||
1) https://www.ncbi.nlm.nih.gov/pubmed/18600226 | 1) https://www.ncbi.nlm.nih.gov/pubmed/18600226 | ||
Revision as of 08:35, 21 October 2019
Plasmid vector for SIGEX screening
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 7
Illegal AgeI site found at 119 - 1000INCOMPATIBLE 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.