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Bacterial Sketch Pad: Exploring Collins Constructs and BioBricks

This group meeting on 2005-07-05 discusses the progress on Collins constructs and BioBricks. Topics cover pairwise assembly advancements, successful ligations, sequencing mutations, and introducing new variants. Participants aim to optimize various genetic constructs for further experimentation. The session includes a detailed talk outline, accomplishments, ongoing experiments, and planned activities within the Collins and BioBricks projects.

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Bacterial Sketch Pad: Exploring Collins Constructs and BioBricks

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Presentation Transcript

  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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