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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18. BioBricks Team. Ligations. Pairwise Assembly Successful Ligations P l + QPI LacIts(241) P l + QPI LacIts(265) P l + RBS-mCherry-T

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18

  2. BioBricks Team

  3. Ligations • Pairwise Assembly • Successful Ligations • Pl+ QPI LacIts(241) • Pl+ QPI LacIts(265) • Pl + RBS-mCherry-T • PlacI + RBS-mCherry-T • PlacI-Hyb + RBS-mCherry-T • P434 + RBS-mCherry-T • PlacI-Hyb + RBS-Venus-T • Pl + RBS-Venus-T • PlacI-Hyb +RBS-lcI • Unsuccessful Ligations • PLacIQPIl • PlQPI • QPIlRBS-mCherry-T • QPI434+RBS-mCherry-T • PlacI(hyb) + RBS-lambda • Pl + RBS-434 • Pl + QPILacI • PlacI + RBS-Venus-T • P434 + RBS-Venus-T

  4. Lambda cI mutations • Made two variants of temperature sensitive Lambda cI, they will be sequenced this week and incorporated into the ligation schedule.

  5. Bacterial mCherry BioBrick • Removed internal PstI site from bacterial mCherry • Added BioBrick ends • Two versions: with or w/o LVA degradation tag • Will miniprep and send for sequencing today

  6. This week with Biobricks • Pairwise Assembly • Terminator + P434-mCherry • Terminator + P434-Venus • Terminator + Pl-mCherry • Terminator + Pl-Venus • Repeat all unsuccessful ligations from last week

  7. CollinsMod Team

  8. What has been done: CollinsMod • Cloning mCherry and GFP reporter constructs, as well as the empty vector pTV • Testing the Collins Circuit • Replicating the Kobayashi work exactly

  9. PL* mCherry pWCh Cloning CI-Repressed mCherry (pWCh) • Bacteria-optimized mCherry cloned successfully, verified by: • Analytical digests • Cells spun down are visibly pink/red/violet • Cells (on plate & in-solution) fluorescece when excited with ~600nm (bleaches very quickly) • Sequences pending • Transformants that were streaked out were not visibly "cherry" until at least two days later, and even then not as darkly colored as expected/desired.

  10. Ptrc gfpmut3b pEG Other Cloning Experiments • Cloning pTV, the backbone of the toggle-switch vector lacking lacI or cI • Analytical digests indicate correct fragments • Co-transformation with the reporter constructors will indicate whether the presence of the vector affects fluorescence • Cloning of pEG, the LacI-repressed GFP reporter • Analytical digests indicate correct fragments

  11. pWG PL* gfpmut3b pTS Ptrc l cI lacI PL* Testing the Collins Circuit • Parental strain • MC4100 (lacI-; from The Registry) • Introduced constructs • cI-repressed GFP reporter (pWG), alone • reporter (pWG) + toggle-switch (pTS) • Test conditions • 0mM IPTG • 0, 12, 24, 48, 96, 192, 284J/m2 UV • Assay time • next day

  12. 384J/m2 UV kills most cells

  13. Circuit Response to UV (I) 0 J/m2 reporter-only (pWG) 0 J/m2 12 J/m2 24 J/m2 48 J/m2 on plate MC4100 background 10x objective FITC 96 J/m2 192 J/m2 384 J/m2 reporter (pWG) + toggle switch (pTS)

  14. 12 J/m2 24 J/m2 48 J/m2 0 J/m2 96 J/m2 192 J/m2 384 J/m2 Consitutive GFP Expression Low in solution pWG in MC4100 100x, oil-immersion FITC & DIA-DLL

  15. 12 J/m2 24 J/m2 48 J/m2 0 J/m2 96 J/m2 192 J/m2 384 J/m2 Circuit Response to UV (I) in solution pWG+pTS in MC4100 100x, oil-immersion FITC & DIA-DLL

  16. pWG PL* mCherry pWCh PL* gfpmut3b pTS Ptrc l cI lacI PL* Replicating Kobayashi et al.'s Work Exactly • Strain: JM 2.300 • Used by Kobayashi et al. (2004) • Has been made competent • Introduced Constructs • CI-repressed GFP reporter (pWG)-only • CI-repressed mCherry reporter (pWCh)-only • GFP reporter (pWG) + toggle switch (pTS) • mCherry reporter (pWCh) + toggle switch (pTS) • GFP repoter (pWG) + empty vector (pTV) • mCherry repoter (pWCh) + empty vector (pTV) • Test Conditions • 0, 2mM IPTG • 0, 6, 12, 24, 48 (96, 192, 384?) J/m2 UV • Assay Conditions • 4h and 16h after UV irradiation • Cells will also be examined before and after the addition of IPTG

  17. CollinsMod: A Summary • Cloning experiments (success!) • mCherry reporter, regulated by CI (pWCh) • GFP reporter, regulated by LacI (pEG) • empty toggle-switch vector (pTV) • Testing the Collins circuit • UV kills readily at 384J/m2, and even at 192J/m2. • On-plate assay indicates upregulation of GFP expression following UV treatment • Not clearly corroborated by in-solution, single-cell examination • No IPTG was used • Constitutive GFP expression much lower than expected • Replicating Kobayashi's work exactly • On our way!

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