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

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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

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Lambda cI mutationsLambda cI mutationsMade two variants of temperature sensitive Lambda cI, they will be sequenced this week and incorporated into the ligation schedule.

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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

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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

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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

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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

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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

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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

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384J/m384J/m22 UV kills most cells UV kills most cells

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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

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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

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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

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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

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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!