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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Yves Wang, Jennifer Gao, Yin Li Chris Doucette, Thomas Noriega, Hing Eng Group Meeting 2005-07-05. Talk outline. What has been done with the Collins constructs What has been done with the BioBricks What’s in store for this week. Collins (Yin and Jenny).

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Yves Wang, Jennifer Gao, Yin Li Chris Doucette, Thomas Noriega, Hing Eng Group Meeting 2005-07-05

  2. Talk outline • What has been done with the Collins constructs • What has been done with the BioBricks • What’s in store for this week

  3. Collins (Yin and Jenny)

  4. What has been done with Biobricks • Pairwise assembly: • QPI digestions successful with modified protocol: • Pl + QPILac • QPIl + YFP • QPI434 + YFP • Completed first two rounds and started third round of ligations to build QPIs from scratch • Performing ligations in duplicate to decrease chances of setbacks • Sequenced successful ts (mut265) mutations in LacI • Have sent LacI ts (mut241) and both QPILacI(ts) mutants (m241 and m265) for sequencing • Outsourced 2 different ts, ind- versions of lcI. (“mJP3” based on Japanese patent Genbank #E07700, and “m8572” based on cI857 sequence found in HENDRIX Lambda II book).

  5. Successful Ligations: mCherry + Terminator RBS + 434 RBS + lcI RBS + mCherry-Terminator RBS + Venus-Terminator Digestions ready for ligation: PlambdaCI P434 PlacI PlacI(hybrid) 434 lcI RBS-434 RBS-lcI RBS-mCherry-Terminator RBS-Venus-Terminator QPIlacI QPIl QPI434 Ligations

  6. Summary: What we have done • CollinsMod: (?) • BioBricks: • Pairwise assembly • mCherry + T • RBS + 434 • RBS + lcI • RBS + mCherry-Terminator • RBS + Venus-Terminator • QPI digest problem solved; ligations still in testing • Mutations and Sequencing • We have a sequenced Bb-LacI m265 • We are sequencing LacI m241 and QPI-lacI m241 and m265 • Introducing mJP3 and m8572 into Lambda cI and QPI-Lambda cI

  7. This week with Collins

  8. This week with Biobricks • More Pairwise Assembly • First Round • PlacI(hyb) + RBS-lCI • Pl + RBS-434 • RBS + LacIts(265) • All promoters + mCherry • All promoters + Venus • Second Round • Terminator + P434-mCherry • Terminator + P434-Venus • Terminator + Pl-mCherry • Terminator + Pl-Venus • Sequence cI857 mutations • Test microscopy with Promoter-Reporter constructs • Are mCherry-expressing colonies red enough? • Make Tsien lab bacterial mCherry into BioBrick • QPIl + mCherry • QPI434 + mCherry • Pl + QPIlacI • Pl-QPIlacI + QPIl-mCherry • Pl-QPIlacI + mCherry • PlacI + QPIl-mCherry • Pl + QPI434-mCherry

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