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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Thomas Noriega, Hing Eng, Yves Wang, Jennifer Gao, Yin Li, Chris Doucette Group Meeting 2005-06-20. Switch Designs. Based on constructs from Collins Lab l CI-LacIts

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Thomas Noriega, Hing Eng, Yves Wang, Jennifer Gao, Yin Li, Chris Doucette Group Meeting 2005-06-20

  2. Switch Designs • Based on constructs from Collins Lab • lCI-LacIts • The only difference between our toggle switch plasmid and that in Kobayashi et al. (2004) is the introduction of ts mutation into LacI. • Based on BioBrick constructs • lCI-LacIts • 434-CI857 • We are also considering appending a heat-shock promoter to the OFF switch (lcI or 434) to turn off the system more quickly.

  3. UV PL* mCherry l cI Ptrc PL* lacIts Heat The CollinsMod Switch

  4. pWG: pCIRa pWCI: pCIE Ptrc l cI PL* mCherry pWC: pWG, gfp replaced w/ mCherry PL* gfpmut3b CollinsMod Switch: Writing via Repression • Reporters • OFF Switch

  5. Ptrc gfpmut3b pEG: pCIRa, PL* replaced w/ Ptrc Ptrc mCherry pEC: pEG, gfp replaced w/ mCherry PL* lacI pEL: pTSMa, del Ptrc-cI PL* lacIts pELts: pEL, ts mut in lacI CollinsMod Switch: Erasing via Repression • Reporters • ON Switches

  6. pEGhs: pWG, PL* replaced w/ Phs (heat-shock promoter) Phs gfpmut3b pWG (same as from Writing via Repression) PL* mCherry pWC (same as from Erasing via Repression) pECIhs: pWCI, Ptrc replaced w/ Phs (heat-shock promoter) Phs l cI PL* gfpmut3b CollinsMod Switch: Erasing via Induction • Reporters • OFF Switch

  7. pTSGC: pEG + pWC gfpmut3b Ptrc PL* mCherry pECIhs (same as in Erasing via Induction) Phs l cI pTSts: pTSts, ts mut in lacI pTS: pTSMa l cI l cI Ptrc Ptrc PL* PL* lacIts lacI CollinsMod Switch: Toggle Switch • Reporters • Switches

  8. BioBrick Switches

  9. BioBrick: Aggregate Parts Coding Region RBS Promoter RBS Terminator Reporter Coding Region Terminator Reporter Quad Part Inverter (QPI)

  10. QPIs and Reporters Have Need LacI QPI LacI(ts) QPI Lambda cI QPI Lambda cI857 QPI 434 cI QPI mCherry GFP (mut3b) Venus

  11. Test Constructs Plambda GFP (mut3b) Plambda Lambda cI QPI LacI QPI LacI QPI GFP (mut3b) PLac GFP (mut3b) Lambda cI QPI Plambda GFP (mut3b) Plambda Lambda cI QPI 434 QPI LacI(ts) QPI GFP (mut3b) Plambda GFP (mut3b) LacI(ts) QPI P434 GFP (mut3b) P434 434 QPI 857 QPI 857 QPI GFP (mut3b)

  12. BioBricks Testing

  13. Introduction of TS Mutations • LacIts (Chao et al., 2002; Yabuta et al., 1995) • Ala241Thr • 100±10 Miller units @ 30° C (induced b-gal activity) • 48000±1080 @ 40° C • Gly265Asp • 210±10 @ 30° C • 42100±815 @ 40° C • Greatest induction @ 37° C • Both are repressed by IPTG • CI857: ? • TS mutations are to be introduced via site-directed mutagenesis for one construct, which can then be subcloned into other constructs. • Alternatively, mutagenesis can be done for all necessary constructs, depending on time and cost considerations. Yabuta et al. 1995. J Biotechnol 39: 67-73; Chao et al. 2002. Biotechnol Bioeng 79: 1-8.

  14. Things We Need to Find Out • Input Parameters: • Optimal UV exposure to switch state • Optimal IPTG concentration to switch state • Optimal heat exposure to switch state • Amount of time a given state must be maintained • Effects of different heat-shock promoters • Other Parameters: • Effects of cell densities/growth phases • Effects of competing inputs • Delay between input and output • Experimental Setup: • How to apply the UV and the IPTG? • How long after seeding lawn to begin testing? • Should we also test in liquid medium? • Does SOS work in stationary phase • Effectiveness of UV laser pointer • Protocol for cotransforming bacteria w. multiple plasmids

  15. Schedule: Week 1 • CollinsMod: • Reconstitute Collins circuit • Introduce TS mut into lacI of pTS • Build mCherry reporter pWC • Research into suitable heat-shock promoters • BioBricks: • Pairwise assembly • RBS + 857 • T + Pl • mCherry + T • Venus + T • Pl + QPILacI • QPIl + GFP • QPI434 + GFP • Introduce TS mut into lacI QPI

  16. Schedule: Week 2 • CollinsMod: • 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 • 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 TS mutations in LacI QPI

  17. 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

  18. 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)

  19. 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

  20. 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)

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