biosketchharvard igem 2005 1 biobrick: aggregate parts quad part inverter (qpi) rbs codingregion...

BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005

11

BioBrick: Aggregate PartsBioBrick: Aggregate Parts

Quad Part Inverter (QPI)Quad Part Inverter (QPI)

RBSRBSCodingCodingRegionRegion

TerminatorTerminator

PromoterPromoter RBSRBS TerminatorTerminator

ReporterReporterCodingCodingRegionRegion

ReporterReporter


22

QPIs and ReportersQPIs and Reporters

LacI QPILacI QPI

GFP (mut3b)GFP (mut3b)

Lambda cI QPILambda cI QPI

434 cI QPI434 cI QPI

HaveHave

LacI(ts) QPILacI(ts) QPI

VenusVenus

Lambda cI857 QPILambda cI857 QPI

NeedNeed

mCherrymCherry


33

Test ConstructsTest ConstructsGFP (mut3b)GFP (mut3b)

LacI QPILacI QPI




434 QPI434 QPI

PPlambdalambda

PPlambdalambda

PPLacLac




857 QPI857 QPI

PP434434

PPlambdalambda

LacI QPILacI QPI

PPlambdalambda



PPlambdalambda




PP434434


434 QPI434 QPI

857 QPI857 QPI


44

BioBricks TestingBioBricks TestingConditioConditionn

Temp (Temp (°° C) C) 3030 3030 3030 3030 4040 4040 4040 4040

IPTGIPTG (-)(-) (-)(-) (+)(+) (+)(+) (-)(-) (-)(-) (+)(+) (+)(+)

UVUV (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+)

QPIsQPIs LacILacI (-)(-) (-)(-) (+)(+) (+)(+) (-)(-) (-)(-) (+)(+) (+)(+)

lambdalambda (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+)

434434 (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+)

857857 (-)(-) (-)(-) (-)(-) (-)(-) (+)(+) (+)(+) (+)(+) (+)(+)

LacI(ts) LacI(ts) (-)(-) (-)(-) (+)(+) (+)(+) (+)(+) (+)(+) (+)(+) (+)(+)

ConditioConditionn

StimulusStimulus UVUV heatheat IPTGIPTG nonenone

Initial StateInitial State (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+) (-)(-) (+)(+)

Toggle Toggle SwitcheSwitchess

LacI (Collins)LacI (Collins) (+)(+) (+)(+) (-)(-) (+)(+) (-)(-) (-)(-) (-)(-) (+)(+)

857857 (+)(+) (+)(+) (-)(-) (-)(-) (-)(-) (+)(+) (-)(-) (+)(+)

LacI(ts) LacI(ts) (+)(+) (+)(+) (-)(-) (-)(-) (-)(-) (-)(-) (-)(-) (+)(+)


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Things We Want to KnowThings We Want to KnowMinimal exposure time to UV to switch stateMinimal concentration of IPTG to switch stateMinimal exposure time to heat to switch stateAmount of time a given state is maintainedTime delay between input and reportingEffect of varying cell densitiesEffect of competing inputsDoes SOS work in stationary phaseEffectiveness of UV laser pointer


66

Schedule - Week 1Schedule - Week 1Week 1

Mutation

Begin introducing ts mutation into LacI coding region and LacI QPI

BioBrick Assembly

RBS + 857 T + Plambda

mCherry + T Venus + TPlambda + QPILacI QPIlambda + GFP

QPI434 + GFP

Collins


77

Schedule - Week 2Schedule - Week 2Week 2

Mutation

Continue mutations

BioBrick Assembly

RBS-857 + T-Plambda (QPI857)

Plambda-QPILac + QPIlambda-GFP

Plambda-QPILac + GFP

Plac + QPIlambda-GFP

Plambda + QPI434-GFP

RBS + mCherry-T RBS + Venus-T

Collins


88

Schedule - Week 3Schedule - Week 3Week 3 Mutation Sequence mutations

BioBrick Testing

Plambda-QPILac-GFP

Plac-QPIlambda-GFP

Plambda-QPI434-GFP

Plambda-QPILac-QPIlambda-GFP (Collins)

BioBrick Assembly

P434 + QPI857

Collins


99

Schedule - Week 4Schedule - Week 4Week 4 BioBrick

Testing IIPlambda-QPILac-GFP

Plac-QPIlambda-GFP

Plambda-QPI434-GFP

Plambda-QPILac-QPIlambda-GFP (Collins)

BioBrick Assembly

P434-QPI857 + GFP

P434-QPI857 + QPI434-GFP

Plambda + QPILac(ts)

Collins


1010


TestingP434-QPI857-GFP

P434-QPI857-QPI434-GFP (857 system)

BioBrick Assembly

Plambda-QPILac(ts) + GFP

Plambda-QPILac(ts) + QPIlambda-GFP

Collins


1111


Testing IIP434-QPI857-GFP

P434-QPI857-QPI434-GFP (857 system)

BioBrick Testing

Plambda-QPILac(ts)-GFP

Plambda-QPILac(ts)-QPIlambda-GFP (Lac(ts) system)

Collins


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Schedule - Week 7+Schedule - Week 7+Padding

biosketchharvard igem 2005 1 biobrick: aggregate parts quad part inverter (qpi) rbs codingregion...

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