BioSketchBioSketchThe bacterial sketch pad.The bacterial sketch pad.

Hing Eng, Yves Wang, Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega

Group Meeting2005-06-27

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Talk outlineTalk outlineWhat has been done with the Collins constructsWhat has been done with the BioBricksWhat’s in store for this week…the weeks followingUseful lessons learned so far

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What has been done with What has been done with the Collinsthe CollinsJenny, and Yin, the floor is yours.

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What has been done with What has been done with the Biobricksthe Biobricks

Received and made minipreps of all of our indispensable parts (some are missing, but we can get around that)Started the first round of ligations necessary to construct our test constructsStarted the first set of base-pair mutations necessary to make LacI and Lambda cI Temperature sensitive.

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LigationsLigationsChris, Yves… go crazy

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LacI MutationsLacI MutationsTwo different point mutations have been introduced into our LacI constructs using the Invitrogen QuiKChangeII kit. We plan to test the two different mutants to see which performs better in our test and final constructs. The Bb_LacI potential mutants are mini-prepped and ready to be sent out for sequencing today.The Bb_QPI-LacI has not been successfully grown after the mutagenesis, the minipreps have been debugged (?) and are ready to be mutated today.

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Lambda cI mutationsLambda cI mutationsThree simultaneous point mutations are going to be introduced into the Lambda cI Biobricks using the Invitrogen Multi-Quikchange kit. The Bb_Lambda cI and Bb_QPI-Lambda cI minipreps are ready to be sent to the Silver lab to be mutated.

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Schedule for the Week (1) Schedule for the Week (1) that has passedthat has passedCheck this to see if what I say is true and fill out what I don’t

knowCollinsMod:

Reconstitute Collins circuit (done)Introduce TS mut into lacI of pTS (done)Build mCherry reporter pWC (?)

BioBricks:Pairwise assembly

RBS + 857 (Bb_857 does not exist, we will make it)T + P(Done)mCherry + T (not done)Venus + T (done)P + QPILacI (?)

QPI + GFP (?)QPI434 + GFP (?)

Introduce TS mut into lacI (One third done)

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This week (2)This week (2)CollinsMod: (Jenny, Yin… any changes?)

Finish pWC and build GFP-mCherry reporter pTSGCTest Collins circuit w. original and modified experimental setupsSequence TS mutations in pTStsTry to get heat-shock promoters

BioBricks:Pairwise assembly (Chris, Yves… changes?)

RBS-857 + T-P (QPI857)

P-QPILac + QPI-GFP

P-QPILac + GFP

Plac + QPI-GFP

P + QPI434-GFPRBS + mCherry-TRBS + Venus-T

Sequence successful mutations in LacI and continue with unsuccessful onesIntroduce mutations into Lambda cI

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SHOULD we stop here with SHOULD we stop here with the schedule?the schedule?During the last presentation we skipped this

part, after all it is only really interesting to us.However if you want to modify the slides that follow please do so and we’ll include them.

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Schedule: Week 3Schedule: Week 3CollinsMod:

Begin trial run of CollinsMod circuit w. pTSts and pWG Test the constructs for Writing via Repression

pWG, pWC, pWCIParameters for UV induction

Build constructs for Erasing via RepressionpEG, pEC, pEL, pELtsParameters for heat induction

BioBricks:Testing

P-QPILac-GFP

Plac-QPI-GFP

Plambda-QPI434-GFP

Plambda-QPILac-QPI-GFP (Collins)

AssemblyP434 + QPI857

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Schedule: Week 4Schedule: Week 4CollinsMod:

Continue testing Writing via RepressionFinish building constructs/begin testing Erasing via RepressionBegin building

BioBricks:Testing

Plambda-QPILac-GFP

Plac-QPIlambda-GFP

Plambda-QPI434-GFP

Plambda-QPILac-QPIlambda-GFP (Collins)

AssemblyP434-QPI857 + GFP

P434-QPI857 + QPI434-GFP

Plambda + QPILac(ts)

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Schedule: Week 5Schedule: Week 5CollinsMod:

Finish testing Erasing via RepressionRe-run CollinsMod circuit w. pTSts & pTSGCBuild constructs for Erasing via Induction

pEGhs, pECIhs

BioBricks:Testing

P434-QPI857-GFP

P434-QPI857-QPI434-GFP (857 system)

AssemblyPlambda-QPILac(ts) + GFP

Plambda-QPILac(ts) + QPIlambda-GFP

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Schedule: Week 6+Schedule: Week 6+CollinsMod:

Fiddle w. the CollinsMod circuit until it worksIntroduce the Erasing via Induction if necessary

BioBricks:Testing

P434-QPI857-GFP

P434-QPI857-QPI434-GFP (857 system)

AssemblyPlambda-QPILac(ts)-GFP

Plambda-QPILac(ts)-QPIlambda-GFP (Lac(ts) system)

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Lessons LearnedLessons LearnedTest your Minipreps rigorously and wisely.The Supergroups, Hypergroups, groups, hypogroups, subgroups and minigroups must all talk, frequently and clearly. Realize that the schedules will be changing constantly, and make the most out of it.…