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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Hing Eng, Yves Wang, Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega Group Meeting 2005-06-27. 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

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Hing Eng, Yves Wang, Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega Group Meeting 2005-06-27

  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 • …the weeks following • Useful lessons learned so far

  3. What has been done with the Collins • Jenny, and Yin, the floor is yours.

  4. 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.

  5. Ligations • Chris, Yves… go crazy

  6. 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.

  7. 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.

  8. 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 + 857 (Bb_857 does not exist, we will make it) • T + Pl (Done) • mCherry + T (not done) • Venus + T (done) • Pl + QPILacI (?) • QPIl + GFP (?) • QPI434 + GFP (?) • Introduce TS mut into lacI (One third done)

  9. 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-857 + T-Pl (QPI857) • Pl-QPILac + QPIl-GFP • Pl-QPILac + GFP • Plac + QPIl-GFP • Pl + QPI434-GFP • RBS + mCherry-T • RBS + Venus-T • Sequence successful mutations in LacI and continue with unsuccessful ones • Introduce mutations into Lambda cI

  10. 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.

  11. 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 • Pl-QPILac-GFP • Plac-QPIl-GFP • Plambda-QPI434-GFP • Plambda-QPILac-QPIl-GFP (Collins) • Assembly • P434 + QPI857

  12. Schedule: Week 4 • CollinsMod: • Continue testing Writing via Repression • Finish building constructs/begin testing Erasing via Repression • Begin building • BioBricks: • Testing • Plambda-QPILac-GFP • Plac-QPIlambda-GFP • Plambda-QPI434-GFP • Plambda-QPILac-QPIlambda-GFP (Collins) • Assembly • P434-QPI857 + GFP • P434-QPI857 + QPI434-GFP • Plambda + QPILac(ts)

  13. 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 • P434-QPI857-GFP • P434-QPI857-QPI434-GFP (857 system) • Assembly • Plambda-QPILac(ts) + GFP • Plambda-QPILac(ts) + QPIlambda-GFP

  14. Schedule: Week 6+ • CollinsMod: • Fiddle w. the CollinsMod circuit until it works • Introduce the Erasing via Induction if necessary • BioBricks: • Testing • P434-QPI857-GFP • P434-QPI857-QPI434-GFP (857 system) • Assembly • Plambda-QPILac(ts)-GFP • Plambda-QPILac(ts)-QPIlambda-GFP (Lac(ts) system)

  15. 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. • …

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