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ClpXPiseatingaprotein
!Itcouldbeyours!
ESBS‐Strasbourg
ESBS‐Strasbourg6November2010 Slide2
5900 km
Introduction
ESBS‐Strasbourg
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide3
Introduction
Alight‐controllablespecificproteindegradationsystem
Whyaprotease?
Project
ClpXPproteasefromE.coli
• Proteindegradationisanimportantbutoftenneglectedpartofproteinregulation
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide4
Introduction
Whylight?
Inductiontime Instantly Delayed
Interferencewithhostmetabolism No Yes
Degradationcontrol On/off Onlyon
Costs Cheap Moreexpensive
PhytochromeB/PIFinteractionsystem
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide5
Introduction
Overview
Tagged Protein
Modified Protease
660nm
730nm
Light impulse
Introduction System Results Applications Conclusion
System description
?
?
ESBS‐Strasbourg6November2010 Slide7
• Protein Degradation :
• Light control : • Specificity :
ClpXP
Phytochrome B DAS tag
(E. coli) & (A. thaliana) (Synthetic)
Features
PIFs
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide8
Design
PhyB-ClpX trimer (PhyB-ClpX trimer) dimer =pseudo hexamer
ClpP
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide9
Functionning
Inactive Active
Introduction System Results Applications Conclusion
660 nm
730 nm
ESBS‐Strasbourg6November2010 Slide10
Howtouseit?
PIF3/6 DAS tag λO tag yourprotein
Introduction System Results Applications Conclusion
Results
ESBS‐Strasbourg6November2010 Slide12
• Signalflowmodel
Modeling
Lightdependent
Veryweak
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide13
• Proteindegradationsimulation
ModelingIntroduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide14
• Adjustmentofdefinedproteinconcentrations
ModelingIntroduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide15
• 15biobrickswerecreatedandsenttotheregistry
BiobricksIntroduction System Results Applications Conclusion
PIF3
PIF6
GFPsuperfoldRFC25
LAA‐tag(wildtype)
DAS‐tag(C‐terminal)
λO‐tag(N‐terminal)PhyB908
PhyB642
ClpX
ΔN‐ClpX
[ΔN‐ClpX]³
linker‐[ΔN‐ClpX]³
PhyB908‐L‐[ΔNClpX]³
PhyB642‐L‐[ΔNClpX]³
Application
ESBS‐Strasbourg6November2010 Slide17
DynamicProteinAnalysisIntroduction System Results Applications Conclusion
Theadvantagesofalight‐guidedactivationallowthemodulationof protein concentration with the combined characteristics ofspecificity,hightemporalprecisionandrapidreversibility.
ESBS‐Strasbourg6November2010 Slide18
Controlleddegradation
Controltheproteinconcentrationinthecellsimplybyusingacomputerconnectedtoalightningdevice!
660nm
730nm[Protein]
CellComputer Lightning Regulation
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide19
Thetightcontrol andsequential natureof a light‐controlledflipflopmechanismcouldbeusedforlight‐controlledmultistepsynthesiswhichahugepotentialforindustrialapplications.
MultistepSynthesisIntroduction System Results Applications Conclusion
Activator GEN1
Activator Gen2
Repressor‐Pif‐DAS
Repressor‐Pif‐DAS
Conclusion
ESBS‐Strasbourg6November2010 Slide21
Conclusion
Tagyourproteintocontrolitsdegradation:Your protein
Fuse your protein to the Tagging system
660nm
730nm
Light impulse
Your protein
Fuse your protein to the Tagging system
?
?
Tethering couple
Activation signal
Chemical, Physical… stimulus
Chooseyourowntetheringsystem:
??
Activation signal
Chemical, Physical… stimulus
?
Interaction
Tethering couple
Introduction System Results Applications Conclusion
BBa_K365014
ClpP 2xHeptamer
ClpX Hexamer
Linker
ClpX Hexamer
ClpP 2xHeptamer
Linker
ESBS‐Strasbourg6November2010 Slide22
Conclusion
Thefunctionalityofthesystemwillbetested:‐Quantitativevalues(Vmax,Km…)Modeling‐Qualitativevalues(false+/‐)Robustness
Proteindegradationdevice:
DNA mRNA Protein
Regulation:Gene
Knockout…siRNA…
Proteinregulation
device
NewRegulation
layer
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide23
ConclusionIntroduction System Results Applications Conclusion
Multi step reaction - Bioprocess
Protein dynamics study - Cell signaling
Complex cellular circuits - BioLogic gates
Enz1
Enz2
Enz3
Enz4
Sub1
Prod1
EnzA
Prod2
tagged
tagged
?
?
Light sensor
Chemical sensor
P C FC
P C RC
P1
CA2
GFP
CR2
P2
CA1
(R
FP
)
CR1
CSB™
Acknowledgment&Sponsors
ESBS:• DirectorClaudeKedinger
• Prof.Annie‐PauleSibler• Prof.BrunoChatton
• Prof.EtienneWeiss• Prof.GeorgesOrfanoudakis
• Prof.PeterPhilippsen
Advisor:
• Prof.JacquesHaiech
Instructors:
• Dr.MariaZeniou
• Prof.ChristopheLallement
• Dr.MorganMadec
External:• Dr.StephanKircher
• Prof.WilfriedWeber• Dr.TaniaBaker
TheHumanProtease
ESBS‐Strasbourg
Questions?
ESBS‐Strasbourg6November2010 Slide27
System
• Light control
Introduction Background Results Applications ConclusionHumanPractice
PIF
730nm
660nm
Light impulse
PhyB
ESBS‐Strasbourg6November2010 Slide28
System
• Degradation
Introduction Background Results Applications ConclusionHumanPractice
ClpXP
ESBS‐Strasbourg6November2010 Slide29
System
• Specificity
Introduction Background Results Applications ConclusionHumanPractice
SspB
ESBS‐Strasbourg6November2010 Slide30
Conclusion
BioLogical Gates
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide31
Conclusion
Systemnotentirelytestedyet.
NeedtooptimizethePhyBexpression:‐CodonUsage
‐PromoterandRBSoptimization
AllneededbiobricksandconstructionaresenttotheRegistry:
BBa_K365010 BBa_K365000 BBa_K365011 BBa_K365001
BBa_K365007 BBa_K365014
whole system
PhyB
ClpX Hexamer
ClpP 2xHeptamer
PIF3/6 DAS
Tagging system Tethering system
ClpX Hexamer
ClpP 2xHeptamer
Linker
Introduction System Results Applications Conclusion
ESBS‐Strasbourg6November2010 Slide32
• PIF6‐linker‐GFP– Requirement
• N‐terminalfusionofPIF6‐linkertoGFPshouldnotdecreaseGFPfluorescence
– Observation• 15.5foldincreaseinGFPfluorescenceduetotheN‐terminalfusion
– Whathappened?
CharacterizationIntroduction Background Results Applications ConclusionHumanPractice
ESBS‐Strasbourg6November2010 Slide33
• PIF6‐linker‐GFP
– Possibleexplanation:Multipletranslationinitiations– Prevention:C‐terminalfusionoflinker‐PIF6
CharacterizationIntroduction Background Results Applications ConclusionHumanPractice
GFP
FulllengthPIF6‐linker‐GFP
Shortenedfragments
ESBS‐Strasbourg6November2010 Slide34
• PhytochromeB– FailedexpressionofPhyB
• Triedover4weeks• variousconditions
– differentinducerconcentrations– differenttemperatures– differenttimepointsafterinduction
– differentstrains(W3110,Bl21,Bl21CodonPlus™,Bl21Star™)
– Intentiontocharacterize• AbilityofPhyBtobindtothechromophore(Zincblot)
• Photoreversibility(differencespectroscopy)
CharacterizationIntroduction Background Results Applications ConclusionHumanPractice
ESBS‐Strasbourg6November2010 Slide35
• InteractionPIF6PhytochromeB
• Testofdegradationtags
• Testoftheassembledsystem
FutureworkIntroduction Background Results Applications ConclusionHumanPractice
ESBS‐Strasbourg6November2010 Slide36
HumanPractice
SURVEY:TheperceptionofSyntheticBiologybythescientificcommunityandthepublic
Introduction System Results Applications Conclusion