BioSketch The bacterial sketch pad.

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Presentation transcript:

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)

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).

Ligations Successful Ligations: Digestions ready for ligation: 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

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

This week with Collins

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