BioSketchHarvard iGEM 2005 3 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 434 + 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 434 + RBS-Venus-T
BioSketchHarvard iGEM 2005 4 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 2005 5 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 2005 7 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 2005 8 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 2005 9 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 2005 10 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 2005 15 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 gfpmut3b PL*PL*PL*PL* pWG pTS cI cI P trc PL*PL*PL*PL*lacI mCherry PL*PL*PL*PL* pWCh
BioSketchHarvard iGEM 2005 16 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!