BioSketch The bacterial sketch pad.

BioSketchBioSketchThe bacterial sketch pad.The bacterial sketch pad.

Chris Doucette, Thomas Noriega, Hing EngYves Wang, Jennifer Gao, Yin Li

Group Meeting2005-08-01

BioBricks TeamBioBricks Team

BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005

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mCherry, yeastmCherry, yeastIssues with RBS-mCherry-T

Sent out for sequencing twiceBoth sequences the sameSequence has no relation to mCherryCause unknownAbandoning

Using EYFP for nowmCherry, bacterial should be ready soon


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Pairwise AssemblyPairwise AssemblySuccessful Ligations:

RBS + mCherry (bact)RBS + mCherry (bact) LVAQPI + EYFP

P -QPI + EYFP

P -QPI + EYFP

P + QPI434-EYFP (?)

RBS-mCherry (bact) + T (?)RBS-mCherry (bact) LVA + T (?)PLH + QPI-EYFP

Two versions of final project (we will be sure this afternoon)


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This week with BiobricksThis week with BiobricksMore Pairwise Assembly

Test microscopy with P-QPIts-EYFP constructs

Start playing with final constructs

CollinsMod TeamCollinsMod Team


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What has been done: What has been done: CollinsModCollinsModTesting the circuits

Collins switch & thermo-sensitive circuitsGFP and mCherry as reporters

Using a FACS analyzer

Cloning experimentsPCR cloning of double-reporter


88

Testing the CircuitTesting the CircuitThe (Final) Circuit:

Treatments:IPTG or heat treatment (expected to reduce reporter expression).UV treatment (expected to increase reporter expression).

PPLL** lacItslacIts cIcI PPtrctrc

gfpmut3bgfpmut3bPPLL**

UVUV

HeatHeat


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Expected Results of Expected Results of IPTG/Heat TreatmentIPTG/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.

StrainStrain 0mM IPTG0mM IPTG

30C*30C*2mM IPTG2mM IPTG

30C*30C*0mM IPTG0mM IPTG

40C/37C40C/37C

Collins switch (pTS) + reporterCollins switch (pTS) + reporter +/-+/- --

40C thermo-sensitive switch 40C thermo-sensitive switch (pTS241) + reporter(pTS241) + reporter

+/-+/- -- --

37C thermo-sensitive switch 37C thermo-sensitive switch (pTS265) + reporter(pTS265) + reporter

+/-+/- -- --

GFP reporter (pWG)GFP reporter (pWG) ++ ++ ++

mCherry reporter (pWCh)mCherry reporter (pWCh) ++ ++ ++


1010

0%

20%

40%

60%

80%

100%

1A 1B 2A 2B 3A 3B 5A 5B

Untreated

IPTG treated

Heat treated

Reporters are turned OFF by Reporters are turned OFF by IPTG/heatIPTG/heat

StrainsStrains UntreatedUntreated IPTG treatedIPTG treated Heat treatedHeat treated

IDID GenotypeGenotype CellsCells FluorFluor % Fluor% Fluor CellsCells FluorFluor % Fluor% Fluor CellsCells FluorFluor % Fluor% Fluor

1A1A Collins switch + GFPCollins switch + GFP 555555 2222 3.96%3.96% 188188 33 1.60%1.60%

1B1B Collins switch + Collins switch + mCherrymCherry

698698 113113 16.19%16.19% 315315 33 0.95%0.95%

2A2A 40C switch + GFP40C switch + GFP 113113 3333 29.20%29.20% 313313 1414 4.47%4.47% 250250 99 3.60%3.60%

2B2B 40C switch + mCherry40C switch + mCherry 414414 6363 15.22%15.22% 228228 11 0.44%0.44% 130130 1313 10.00%10.00%

3A3A 37C switch + GFP37C switch + GFP 132132 2626 19.70%19.70% 215215 66 2.79%2.79% 7070 11 1.43%1.43%

3B3B 37C switch + mCherry37C switch + mCherry 876876 188188 21.46%21.46% 283283 22 0.71%0.71% 350350 99 2.57%2.57%

5A5A GFPGFP 505505 490490 97.03%97.03% 8585 8383 97.65%97.65% 7070 6969 98.57%98.57%

5B5B mCherrymCherry 507507 472472 93.10%93.10% 136136 127127 93.39%93.39% 170170 164164 96.47%96.47%


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UV Treatment Should Turn UV Treatment Should Turn ON SwitchesON SwitchesUV 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.


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A FACS Analyzer to Quantify A FACS Analyzer to Quantify ResultsResultsA 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 experimentFluorophore: GFPDensity: ~1 x 107 cells/mlFilter: 0.22um Millipore Millex-GV membraneTentative appointment made for Aug 9.


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Cloning the Double-ReporterCloning the Double-ReporterpTSGC

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.

pTSGCpTSGCgfpmut3bgfpmut3b PPtrctrc mCherrymCherryPPLL**


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This week with CollinsModThis week with CollinsModAssaying 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

GenotypesCollins switch + GFPGFP (+ve)JM 2.300 (-ve)

ConditionsIPTG treatment (0, 2, 5mM)UV treatment (0, 24, 48J/m2)

BioSketch The bacterial sketch pad.

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