MIT iGEM 20092009.igem.org/files/presentation/MIT.pdf · REVERSIBLE PROTEIN LOCALIZATION MIT iGEM...

MITiGEM2009LightSwitchableProteinLocaliza;onSystem

CoryLi,2012,DepartmentofElectricalEngineering&ComputerScienceAlieDooliHle,2011,DepartmentofBiologicalEngineeringBrianRoss,2011,DepartmentofBiologicalEngineering

DominicMcDonald,2010,DepartmentofChemicalEngineering

FASTREVERSIBLEPROTEINLOCALIZATION

MITiGEM2009Project

Protein‐levelcontrol

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Protein‐levelcontrol

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Protein‐levelcontrol

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WhyLight?

•  Fastinduc;on•  Accessible•  Simple

•  Demonstratedtowork

•  Cheap•  Easytoreversevs.chemicals

SystemOverview

SystemOverview

Goal: Development of Localization System

SystemOverview

Goal: Endogenous Production of Required Compounds

Applica;ons

•  Studies–  CellCycle–  ProteinFunc;onalStudies

•  TemporalandSpa;alProteinRegula;on–  Biofilms–  Colonies

•  Advantages–  NoNeedforChemicalAddi;ves– Orthogonal–  EasilytoSequester

PreviousLightSwitchableSystem

Photolocalizer BacterialPhotography

QuailLabPhy‐Pif

VoigtLab

Host S.cerevisiae E.coli S.cerevisiae Mammalian

Usage SelfContained SelfContained ExogenousChemicalsReq.

ExogenousChemicalReq.

Readout ProteinLocaliza;on

Transcrip;on Transcrip;on ProteinLocaliza;on

Time Seconds Minutes Minutes Seconds

BACKGROUND12

PhyB‐PIF3System

13

Pfr

Far Red Light

Red Light

PhytochromesChangeConforma;oninRedandFar‐redlight

Phycocyanobilin (PCB)

Dr. Jasper J. van Thor. “Phytochrome light receptors.” Laboratory of Molecular Biophysics. (2001)

14

Proteininterac;onsbetweenPhyBandPIF3areswitchablebylight

15Nucleus

PIF3

PhyB

G-Box Light-Sensitive Gene

Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

PhyB‐PIF3SysteminPlantsandAlgae

16

PhyB‐PIF3SysteminPlantsandAlgae

Nucleus

PIF3

PhyB

G-Box Light-Sensitive Gene

Red Light

Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

17

PhyB‐PIF3SysteminPlantsandAlgae

Nucleus

PIF3

PhyB

G-Box Light-Sensitive Gene

Pfr

Red Light

Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

18

PhyB‐PIF3SysteminPlantsandAlgae

Nucleus

PhyB

G-Box Light-Sensitive Gene

Pfr PIF3

Red Light

Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

19

PhyB‐PIF3SysteminPlantsandAlgae

G-Box Light-Sensitive Gene

Red Light

PhyB

Pfr PIF3

Nucleus Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

20

PhyB‐PIF3SysteminPlantsandAlgae

G-Box Light-Sensitive Gene

Red Light

PhyB

Pfr PIF3

Circadian Control

Nucleus Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

21

PhyB‐PIF3SysteminPlantsandAlgae

Nucleus

PIF3

G-Box Light-Sensitive Gene

PhyB

Far-red light

Quail, Peter H. "Phytochrome Photosensory Signaling Networks." Nature. (2002).

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PhyB-PIF3 engineered to make artificial gene promoter system

Sae Shimizu-Sato, Enamul Hug, James M. Tepperman, Peter H. Quail, “A Light Switchable Gene Promoter System”, Nature, (2002).

PIF3

PhyB

Protein

Organelle Membrane

Anchor Complex

Traveler Complex

Red Light

Far-Red Light

PCB

LocalizaKonTargeKng

Signal Sequences

Target Terminus

Mps3p

(181 aa) Nuclear Membrane N

Longin

(162 aa) Vacuole N

Ras2p

(38 aa) Plasma Membrane C

Bcl-2

(22 aa) Outer Mitochondrial

Membrane C

Construct Design Motif for the PhyB-PIF3 Anchor-Traveler System

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Method 1: Sequential Cloning

Amplification of PhyB, PIF3, CFP and YFP

PhyBCFP

pRS313

YFPPIF3

pRS313

Digestion and Ligation of PIF3, PhyB into pRS313

Digestion and Ligation of CFP, YFP into pRS313 with either PhyB or PIF3

pRS313 pRS313

PhyB PIF3

Plasmids were Transformed and Sequenced

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Method2:HomologousRecombina;on

ThePlasmidsalongwiththeircorrespondingDNAwillbecloneddirectlyintoyeast.

Visualization of the Anchor Traveler System through Microscopy

•  Sequencing Results

•  CFP/YFP Florescence Visualization via

Microscope

METABOLICENGINEERINGOFPCB30

CurrentSourcesofPCB

•  Standard,chemicallypurifiedfromSpirulina(blue‐greenalgae,cyanobacteria)

•  E.coliExtrac;on

•  Yeastproduc;on(?)

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InconvenienceofSupplemen;ngPCBtoMedia

• LargeamountoforganicsolventsneededtopurifyPCB

• Needstandardchemistryequipmentthatmanylabsdonothave

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Absorp;onspectrumfrompurifiedPCB

Wavelength(nm)

Absorba

nce

680nm

0

0.05

0.1

0.15

0.2

0.25

300 400 500 600 700 800

Absorba

nce

Wavelength(nm)

PCBExtractedfromSpirulina

33Quail Lab, UC-Berkeley

PCBandPφBBiosynthesisPathway

AdaptedfromGAGambeHaandJCLagarias,PNAS98,no.19(September2001)

HO1(cyanobacteria)HY1(plant)

HY2(plant) PcyA(cyanobacteria)

HMX1(yeast)ONLYUNDER

IRONSTARVATION!

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HMX1Over‐Expression

•  PCRHMX1genefromYeastgenome

•  CloneHMX1intoavector

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PCBandPφBBiosynthesisPathway

AdaptedfromGAGambeHaandJCLagarias,PNAS98,no.19(September2001)

HO1(cyanobacteria)HY1(plant)HMX1(yeast)

HY2(plant) PcyA(cyanobacteria)

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PcyA/HY2SynthesisinYeast

•  CodonOp;mizedwithGeneART

•  ClonedPcyAop;mizedintoavector

•  FailedcloningofHY2

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Func;onalAssay

•  NeedamethodtodetermineifPCBisactuallybeingproduced

•  UsedstrainsdevelopedbyQuaillabatUC‐Berkeleytocreateafunc;onalassaytotestforPCB

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Lysecells

Treatw/ONPGandwait

Successfulproduc;onofβ‐galactosidase

(‐)control

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Func;onalAssay

357

11.48

414.5

4.72 4.01 1.24 0.07 0.31 0

50

100

150

200

250

300

350

400

450

B-G

al E

xpre

ssio

n (M

iller

Uni

ts)

LacZ Activity

+PCBRedLight

‐PCBRedLight

+PCBDark

‐PCBRedLight

Experiment1

Experiment2

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ProgressovertheSummer

•  Plasmidw/ADH1pdrivingHMX1

•  Plasmidw/ADH1pdrivingPcyA

•  Plasmidw/ADH1pdrivingHY2

•  WorkingFunc;onalAssay

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Integra;onofMetabolicEngineeringofPCBandPhyB‐PIF3Localiza;on

43

Integra;onofMetabolicEngineeringofPCBandPhyB‐PIF3Localiza;on

44

Red Light

Integra;onofMetabolicEngineeringofPCBandPhyB‐PIF3Localiza;on

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Far-red light

Acknowledgements

•  Faculty Advisors –  Narendra Maheshri, Ph. D

Dept. of Chemical Engineering –  Kristala Prather, Ph. D

Dept. of Chemical Engineering –  Natalie Kuldell, Ph. D

Dept. of Biological Engineering

Quail Laboratory – UC-Berkeley

•  Graduate Advisors –  Brian Belmont

Dept. of Biological Engineering –  Chia-Yung Wu

Dept. of Biology –  Jeffrey Wagner

Dept. of Biological Engineering –  Nathan Klapoetke

Dept. of Biological Engineering –  Scott Carlson

Dept. of Biological Engineering –  Stephen Goldfless

Dept. of Biological Engineering –  Shawn Finney-Manchester

Dept. of Chemical Engineering –  T.L. To

Dept. of Chemical Engineering –  Tae Seok Moon

Dept. of Chemical Engineering