mit igem 20092009.igem.org/files/presentation/mit.pdf · reversible protein localization mit igem...
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MITiGEM2009LightSwitchableProteinLocaliza;onSystem
CoryLi,2012,DepartmentofElectricalEngineering&ComputerScienceAlieDooliHle,2011,DepartmentofBiologicalEngineeringBrianRoss,2011,DepartmentofBiologicalEngineering
DominicMcDonald,2010,DepartmentofChemicalEngineering
FASTREVERSIBLEPROTEINLOCALIZATION
MITiGEM2009Project
Protein‐levelcontrol
SwitchOff
Protein‐levelcontrol
SwitchOn
Protein‐levelcontrol
SwitchOff
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).
22
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
26
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
28
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(?)
31
InconvenienceofSupplemen;ngPCBtoMedia
• LargeamountoforganicsolventsneededtopurifyPCB
• Needstandardchemistryequipmentthatmanylabsdonothave
32
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
35
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
37
Func;onalAssay
• NeedamethodtodetermineifPCBisactuallybeingproduced
• UsedstrainsdevelopedbyQuaillabatUC‐Berkeleytocreateafunc;onalassaytotestforPCB
38
Lysecells
Treatw/ONPGandwait
Successfulproduc;onofβ‐galactosidase
(‐)control
39
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
41
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
45
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