BioSketch BioSketch The bacterial sketch pad. The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18
Jan 11, 2016
BioSketchBioSketchThe bacterial sketch pad.The bacterial sketch pad.
Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang
Group Meeting2005-07-18
BioBricks TeamBioBricks Team
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
33
LigationsLigationsPairwise Assembly
Successful LigationsP + QPI LacIts(241)
P + QPI LacIts(265)
P + RBS-mCherry-T
PlacI + RBS-mCherry-T
PlacI-Hyb + RBS-mCherry-T
P434 + RBS-mCherry-T
PlacI-Hyb + RBS-Venus-T
P + RBS-Venus-T
PlacI-Hyb +RBS-cIUnsuccessful Ligations
PLacIQPIPQPIQPIRBS-mCherry-T
QPI434+RBS-mCherry-T
PlacI(hyb) + RBS-lambda
P + RBS-434
P + QPILacI
PlacI + RBS-Venus-T
P434 + RBS-Venus-T
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
44
Lambda cI mutationsLambda cI mutationsMade two variants of temperature sensitive Lambda cI, they will be sequenced this week and incorporated into the ligation schedule.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
55
Bacterial mCherry BioBrickBacterial mCherry BioBrickRemoved internal PstI site from bacterial mCherryAdded BioBrick endsTwo versions: with or w/o LVA degradation tagWill miniprep and send for sequencing today
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
66
This week with BiobricksThis week with BiobricksPairwise Assembly
Terminator + P434-mCherry
Terminator + P434-Venus
Terminator + P -mCherry
Terminator + P -Venus
Repeat all unsuccessful ligations from last week
CollinsMod TeamCollinsMod Team
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
88
What has been done: What has been done: CollinsModCollinsModCloning mCherry and GFP reporter
constructs, as well as the empty vector pTV
Testing the Collins Circuit
Replicating the Kobayashi work exactly
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
99
Cloning CI-Repressed Cloning CI-Repressed mCherry (pWCh)mCherry (pWCh)
Bacteria-optimized mCherry cloned successfully, verified by:
Analytical digestsCells spun down are visibly pink/red/violetCells (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.
mCherrymCherryPPLL** pWChpWCh
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1010
Other Cloning ExperimentsOther Cloning ExperimentsCloning pTV, the backbone of the toggle-switch vector lacking lacI or cI
Analytical digests indicate correct fragmentsCo-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 fragmentsgfpmut3bgfpmut3b pEGpEGPPtrctrc
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1111
Testing the Collins CircuitTesting the Collins CircuitParental strain
MC4100 (lacI-; from The Registry)
Introduced constructscI-repressed GFP reporter (pWG), alonereporter (pWG) + toggle-switch (pTS)
Test conditions0mM IPTG0, 12, 24, 48, 96, 192, 284J/m2 UV
Assay timenext day
gfpmut3bgfpmut3bPPLL** pWGpWG pTSpTS cIcIPPtrctrcPPLL**lacIlacI
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1212
384J/m384J/m22 UV kills most cells UV kills most cells
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1313
Circuit Response to UV (I)Circuit Response to UV (I)
reporter-only (pWG)reporter-only (pWG)
reporter (pWG) + toggle switch (pTS)reporter (pWG) + toggle switch (pTS)
0 J/m2
0 J/m2 12 J/m2 24 J/m2 48 J/m2
96 J/m2 192 J/m2 384 J/m2
on plateon plateMC4100 backgroundMC4100 background10x objective10x objectiveFITCFITC
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1414
Consitutive GFP Expression Consitutive GFP Expression Low Low
0 J/m20 J/m2 12 J/m212 J/m2 24 J/m224 J/m2 48 J/m248 J/m2
192 J/m2192 J/m296 J/m296 J/m2 384 J/m2384 J/m2
in solutionin solutionpWG in MC4100pWG in MC4100100x, oil-immersion100x, oil-immersionFITC & DIA-DLLFITC & DIA-DLL
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1515
Circuit Response to UV (I)Circuit Response to UV (I)
0 J/m20 J/m2 12 J/m212 J/m2 24 J/m224 J/m2 48 J/m248 J/m2
192 J/m2192 J/m296 J/m296 J/m2 384 J/m2384 J/m2
in solutionin solutionpWG+pTS in MC4100pWG+pTS in MC4100100x, oil-immersion100x, oil-immersionFITC & DIA-DLLFITC & DIA-DLL
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1616
Replicating Kobayashi et Replicating Kobayashi et al.'s Work Exactlyal.'s Work ExactlyStrain: JM 2.300
Used by Kobayashi et al. (2004)Has been made competent
Introduced ConstructsCI-repressed GFP reporter (pWG)-onlyCI-repressed mCherry reporter (pWCh)-onlyGFP reporter (pWG) + toggle switch (pTS)mCherry reporter (pWCh) + toggle switch (pTS)GFP repoter (pWG) + empty vector (pTV)mCherry repoter (pWCh) + empty vector (pTV)
Test Conditions0, 2mM IPTG0, 6, 12, 24, 48 (96, 192, 384?) J/m2 UV
Assay Conditions4h and 16h after UV irradiationCells will also be examined before and after the addition of IPTG
gfpmut3bgfpmut3bPPLL** pWGpWG
pTSpTS cIcIPPtrctrcPPLL**lacIlacI
mCherrymCherryPPLL** pWChpWCh
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1717
CollinsMod: A SummaryCollinsMod: A SummaryCloning experiments (success!)
mCherry reporter, regulated by CI (pWCh)GFP reporter, regulated by LacI (pEG)empty toggle-switch vector (pTV)
Testing the Collins circuitUV 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 examinationNo IPTG was used
Constitutive GFP expression much lower than expected
Replicating Kobayashi's work exactlyOn our way!