BioSketch The bacterial sketch pad. Hing Eng, Yves Wang, Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega Group Meeting

BioSketchHarvard iGEM Talk outline What has been done with the Collins constructs What has been done with the BioBricks What’s in store for this week …the weeks following Useful lessons learned so far

BioSketchHarvard iGEM What has been done with the Collins Jenny, and Yin, the floor is yours.

BioSketchHarvard iGEM What has been done with the 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 constructs Started the first set of base-pair mutations necessary to make LacI and Lambda cI Temperature sensitive.

BioSketchHarvard iGEM Ligations Chris, Yves… go crazy

BioSketchHarvard iGEM LacI Mutations Two 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.

BioSketchHarvard iGEM Lambda cI mutations Three 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.

BioSketchHarvard iGEM Schedule for the Week (1) that has passed Check this to see if what I say is true and fill out what I don’t know CollinsMod: Reconstitute Collins circuit (done) Introduce TS mut into lacI of pTS (done) Build mCherry reporter pWC (?) BioBricks: Pairwise assembly RBS (Bb_857 does not exist, we will make it) T + P  (Done) mCherry + T (not done) Venus + T (done) P + QPI LacI (?) QPI + GFP (?) QPI GFP (?) Introduce TS mut into lacI (One third done)

BioSketchHarvard iGEM This week (2) CollinsMod: (Jenny, Yin… any changes?) Finish pWC and build GFP-mCherry reporter pTSGC Test Collins circuit w. original and modified experimental setups Sequence TS mutations in pTSts Try to get heat-shock promoters BioBricks: Pairwise assembly (Chris, Yves… changes?) RBS T-P (QPI 857 ) P -QPI Lac + QPI -GFP P -QPI Lac + GFP P lac + QPI -GFP P + QPI 434 -GFP RBS + mCherry-T RBS + Venus-T Sequence successful mutations in LacI and continue with unsuccessful ones Introduce mutations into Lambda cI

BioSketchHarvard iGEM SHOULD we stop here with 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.

BioSketchHarvard iGEM Schedule: Week 3 CollinsMod: Begin trial run of CollinsMod circuit w. pTSts and pWG Test the constructs for Writing via Repression pWG, pWC, pWCI Parameters for UV induction Build constructs for Erasing via Repression pEG, pEC, pEL, pELts Parameters for heat induction BioBricks: Testing P -QPI Lac -GFP P lac -QPI -GFP P lambda -QPI 434 -GFP P lambda -QPI Lac -QPI -GFP (Collins) Assembly P QPI 857

BioSketchHarvard iGEM Schedule: Week 4 CollinsMod: Continue testing Writing via Repression Finish building constructs/begin testing Erasing via Repression Begin building BioBricks: Testing P lambda -QPI Lac -GFP P lac -QPI lambda -GFP P lambda -QPI 434 -GFP P lambda -QPI Lac -QPI lambda -GFP (Collins) Assembly P 434 -QPI GFP P 434 -QPI QPI 434 -GFP P lambda + QPI Lac(ts)

BioSketchHarvard iGEM Schedule: Week 5 CollinsMod: Finish testing Erasing via Repression Re-run CollinsMod circuit w. pTSts & pTSGC Build constructs for Erasing via Induction pEGhs, pECIhs BioBricks: Testing P 434 -QPI 857 -GFP P 434 -QPI 857 -QPI 434 -GFP (857 system) Assembly P lambda -QPI Lac(ts) + GFP P lambda -QPI Lac(ts) + QPI lambda -GFP

BioSketchHarvard iGEM Schedule: Week 6+ CollinsMod: Fiddle w. the CollinsMod circuit until it works Introduce the Erasing via Induction if necessary BioBricks: Testing P 434 -QPI 857 -GF P P 434 -QPI 857 -QP I 434 -GFP (857 system) Assembly P lambda -QPI Lac(ts) -GFP P lambda -QPI Lac(ts) -QPI lambda -GFP (Lac(ts) system)

BioSketchHarvard iGEM Lessons Learned Test 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. …