Presentation on theme: "BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18."— Presentation transcript:
BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting
BioSketchHarvard iGEM Ligations Pairwise Assembly Successful Ligations P + QPI LacIts(241) P + QPI LacIts(265) P + RBS-mCherry-T P lacI + RBS-mCherry-T P lacI-Hyb + RBS-mCherry-T P RBS-mCherry-T P lacI-Hyb + RBS-Venus-T P + RBS-Venus-T P lacI-Hyb +RBS- cI Unsuccessful Ligations P LacI QPI P QPI QPI RBS-mCherry-T QPI 434 +RBS-mCherry-T P lacI(hyb) + RBS-lambda P + RBS-434 P + QPI LacI P lacI + RBS-Venus-T P RBS-Venus-T
BioSketchHarvard iGEM Lambda cI mutations Made two variants of temperature sensitive Lambda cI, they will be sequenced this week and incorporated into the ligation schedule.
BioSketchHarvard iGEM This week with Biobricks Pairwise Assembly Terminator + P 434 -mCherry Terminator + P 434 -Venus Terminator + P -mCherry Terminator + P -Venus Repeat all unsuccessful ligations from last week
BioSketchHarvard iGEM 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
BioSketchHarvard iGEM 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. mCherry PL*PL*PL*PL* pWCh
BioSketchHarvard iGEM 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 gfpmut3bpEG P trc
BioSketchHarvard iGEM 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/m 2 UV Assay time next day gfpmut3b PL*PL*PL*PL* pWG pTS cI cI P trc PL*PL*PL*PL*lacI
BioSketchHarvard iGEM J/m 2 UV kills most cells
BioSketchHarvard iGEM Circuit Response to UV (I) 0 J/m2 12 J/m2 24 J/m2 48 J/m2 192 J/m2 96 J/m2 384 J/m2 in solution pWG+pTS in MC x, oil-immersion FITC & DIA-DLL
BioSketchHarvard iGEM Replicating Kobayashi et al.'s Work Exactly Strain: JM 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 gfpmut3b PL*PL*PL*PL* pWG pTS cI cI P trc PL*PL*PL*PL*lacI mCherry PL*PL*PL*PL* pWCh
BioSketchHarvard iGEM 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!