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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-08-01. BioBricks Team. Issues with RBS-mCherry-T Sent out for sequencing twice Both sequences the same Sequence has no relation to mCherry Cause unknown

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-08-01

  2. BioBricks Team

  3. Issues with RBS-mCherry-T Sent out for sequencing twice Both sequences the same Sequence has no relation to mCherry Cause unknown Abandoning Using EYFP for now mCherry, bacterial should be ready soon mCherry, yeast

  4. Successful Ligations: RBS + mCherry (bact) RBS + mCherry (bact) LVA QPI434 + EYFP Pl -QPI265 + EYFP Pl -QPI241 + EYFP Pl + QPI434-EYFP (?) RBS-mCherry (bact) + T (?) RBS-mCherry (bact) LVA + T (?) PLH + QPIl-EYFP Two versions of final project (we will be sure this afternoon) Pairwise Assembly

  5. This week with Biobricks • More Pairwise Assembly • Test microscopy with Pl-QPIts-EYFP constructs • Start playing with final constructs

  6. CollinsMod Team

  7. What has been done: CollinsMod • Testing the circuits • Collins switch & thermo-sensitive circuits • GFP and mCherry as reporters • Using a FACS analyzer • Cloning experiments • PCR cloning of double-reporter

  8. UV PL* gfpmut3b l cI Ptrc PL* lacIts Heat Testing the Circuit • The (Final) Circuit: • Treatments: • IPTG or heat treatment (expected to reduce reporter expression). • UV treatment (expected to increase reporter expression).

  9. Expected Results of IPTG/Heat Treatment • Collins switch was always tested @ 37C. • The switch on pTS241 is (supposed to be) turned off @ 40C. • The switch on pTS265 is (supposed to be) turned off @ 37C. • Bacterial mCherry and GFP were both used as reporters.

  10. Reporters are turned OFF by IPTG/heat

  11. UV Treatment Should Turn ON Switches • UV intensities used • 0, 6, 12, 24, 48J/m2 • Plated- and UV-treated cells were allowed to grow to confluence, which took two nights @ 30C. • Results • Inconclusive: There was no visible difference between UV-treated and untreated cells.

  12. A FACS Analyzer to Quantify Results • A FACS analyzer will permit rapid and high-resolution quantification of results. • Experiment planned for FACS analyzer @ Dana Farber • Spoke with the flow cytometry expert @ the Bauer Center • ~12 samples for pilot experiment • Fluorophore: GFP • Density: ~1 x 107 cells/ml • Filter: 0.22um Millipore Millex-GV membrane • Tentative appointment made for Aug 9.

  13. pTSGC gfpmut3b Ptrc PL* mCherry Cloning the Double-Reporter • pTSGC • GFP is repressed by LacI (and turned ON by IPTG/heat) • mCherry is repressed by CI (and turned ON by UV) • Sequencing for P(trc)-GFP plasmid is pending. • PCR has been successful in preparing P(L*)-mCherry for insertion into the already-made P(trc)-GFP plasmid.

  14. This week with CollinsMod • Assaying GFP expression with a FACS analyzer • Current plan: Stagger several experiments so that the following parameters can be examined in parallel: • IPTG treatment (16h and 2 days later) • Heat treatment (16h and 2 days later) • UV treatment (4h and 16h later) • Alternatively: Scale down the parameters for pilot experiment with FACS analyzer • Genotypes • Collins switch + GFP • GFP (+ve) • JM 2.300 (-ve) • Conditions • IPTG treatment (0, 2, 5mM) • UV treatment (0, 24, 48J/m2)

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