BioSketchBioSketchThe bacterial sketch pad.The bacterial sketch pad.

Yves Wang, Jennifer Gao, Yin LiChris Doucette, Thomas Noriega, Hing Eng

Group Meeting2005-07-05

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Talk outlineTalk outline

What has been done with the Collins constructsWhat has been done with the BioBricksWhat’s in store for this week

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Collins (Yin and Jenny)Collins (Yin and Jenny)

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What has been done with What has been done with BiobricksBiobricks

Pairwise assembly:QPI digestions successful with modified protocol:

P + QPILac

QPI + YFPQPI434 + YFP

Completed first two rounds and started third round of ligations to build QPIs from scratchPerforming ligations in duplicate to decrease chances of setbacks

Sequenced successful ts (mut265) mutations in LacIHave sent LacI ts (mut241) and both QPILacI(ts) mutants (m241 and m265) for sequencingOutsourced 2 different ts, ind- versions of cI. (“mJP3” based on Japanese patent Genbank #E07700, and “m8572” based on cI857 sequence found in HENDRIX Lambda II book).

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LigationsLigationsSuccessful Ligations:

mCherry + TerminatorRBS + 434RBS + cIRBS + mCherry-TerminatorRBS + Venus-Terminator

Digestions ready for ligation:

PlambdaCI

P434

PlacI

PlacI(hybrid)

434cI

RBS-434RBS-cIRBS-mCherry-TerminatorRBS-Venus-TerminatorQPIlacI

QPIQPI434

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Summary: What we have Summary: What we have donedoneCollinsMod: (?)

BioBricks:Pairwise assembly

mCherry + TRBS + 434RBS + cIRBS + mCherry-TerminatorRBS + Venus-Terminator

QPI digest problem solved; ligations still in testingMutations and Sequencing

We have a sequenced Bb-LacI m265We are sequencing LacI m241 and QPI-lacI m241 and m265Introducing mJP3 and m8572 into Lambda cI and QPI-Lambda cI

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This week with CollinsThis week with Collins

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This week with BiobricksThis week with BiobricksMore Pairwise Assembly

First RoundPlacI(hyb) + RBS-CI

P + RBS-434RBS + LacIts(265)All promoters + mCherryAll promoters + Venus

Second RoundTerminator + P434-mCherry

Terminator + P434-Venus

Terminator + P -mCherry

Terminator + P -VenusSequence cI857 mutations

Test microscopy with Promoter-Reporter constructsAre mCherry-expressing colonies red enough?

Make Tsien lab bacterial mCherry into BioBrick

QPI + mCherry

QPI + mCherry

P + QPIlacI

P-QPIlacI + QPI-mCherry

P-QPIlacI + mCherry

PlacI + QPI-mCherry

P + QPI434-mCherry