Intelligent sun protection
Transcript of Intelligent sun protection
UVR8 - plant photoreceptor
Intelligent sun protection
Achievements
Dark: Dimer UV: Monomer
Dire
ct U
V de
tect
ion
Indi
rect
UV
dete
ctio
n
Fusion with TetRDBD
Biology Modeling
ETH Zurich, SwitzerlandIsaak Müller, Lisa Seyfarth, Gintas Vainorius, Deborah Huber, Sandro Kundert, Stefan Ganscha, David Seifert, Tim Enke
Advisors: Dr. Johannes Härle, Moritz Lang, Markus JeschekInstructors: Prof. Sven Panke, Prof. Jörg Stelling
Decoder
TetRDBD - a novel two hybrid screening in E.coli
Integration of UVR8 as a transcription factor
Engineering of novel hybrid promoters
Characterization of our BioBricks
We got insight in our system through modeling
Human practices(NRP59)
TetRDBD can not bind as a monomer
UVR8-TetRDBD: A novel transcription factorBinds to the Ptet promoter in absence of UV-B
NOR gate logics: Blue light only: green pigmentRed light only: red pigmentBlue and Red: violet pigment
Team 2012
E.colipse is an intelligent and adaptive sun radiation protection system which responds to UV exposure with the production of the protective agent PABA. Additionally a violet pigment is produced as a warning signal. To achieve this we have developed two detection methods: A direct detection by engineering a novel UV-B sensitive transcription factor and an indirect detection by incorporating two existing photosensors into a decoder.
Verena Jäggin, Daniel Gerngross, Andreas Bosshart, Christian Mayer, Fabian Rudolf, Sonja Billerbeck from the Department of Biosystems Science and Engineering (D-BSSE), ETHZ
Roman Ulm, University of Geneva
Jeff Tabor, Rice University (Houston)
Acknowledgements References Outlook Induce the UVR8-TetRDBD with UV-BTest the hybrid promoters (FACS) and implement them into the decoderBuild up the decoder step by stepMeasure PABA production using HPLC
Photoinduction
Christie J.M. et al, Science, 355 (2012), 1492.Cox R.S. et al, Molecular systems biology, 3 (2007), 145Heijde M. et al, Trends in plant science, 17 (2012), 230Mancinelli A. et al, Plant physiology, 82 (1986), 956Strickland D. et al, PNAS, 105 (2008), 10709
Cph8active
Cph8*inactive
PompC tetR
Red / Intensity
LOVinactive
LOV*active
Ptrp lacI
Blue / UV
k LOVhν
k LOVdecay
KM LOV
lacO cItetO VioletPigment
KM TetR KM LacI
tetO RedPigmentOR
pabAB
kPL
lacO GreenPigmentOR
kPR kPRKM TetRKM cI KM cI KM LacI
kCph8hν
kCph8decay
KM Cph8 kPompC kPtrp
2 UVR8-TetRDBDmonomer
inactive
Ptet pabA pabB
OHO
OH
O
O OH
NH2O
OH
O
O OH
NH2
O OH
pabC
PabAB PabCquasi
steady-state
Chorismic acid 4-amino-4-deoxychorismate(ADC)
4-Aminobenzoic acid(PABA)
UVR8-TetRDBDdimeractive
negative feedback
kUVR8decay
kUVR8hν
KM TetRkPtet
koutKM PabABkcat
UVR8-TetRDBD represses GFP expression
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PABA
No inhibitioninhibition
Red Light Blue Light Output Source
0 0 - darkness
1 0 Red Pigment classical tungsten light bulb
0 1 Green Pigment CCFL
1 1 PABAViolet Pigment sun light
• Blue light receptor: LovTap (Fusion Lov2 and TrpR)• Red light receptor: Cph8 (Fusion Chp1 and EnvZ kinase)• Also inducible with IPTG (”blue light”) and aTc (”red light”) -> proof of principle
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ianc
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sun
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ianc
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ol m
−2]
λ [nm]
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rel.
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ol−1
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λ [nm]
lov−a
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1Absorption spectrum lov−a
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.
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k =λNλ σλ dλ ≈
λ
Nλ σλ ∆ λ
The photoinduction model calculates the activity of light receptors upon light exposure. It takes emission spectra of the light source and absorption spectra, quantum yield and extinction coefficients of the receptors and returns the activation constants for the given light conditions.
Photoinduction
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Green
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RedBlue
RedBlue
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Photoreceptor(dark)
Photoreceptor(light)
Results
Results
kPL < kPR < kPompC < kPtrp
From the decoder model we could conclude the promoter strength of our decoder promoters and use the information to design and implement the biological system.
PABA production is catalyzed by three enzymes PabA, PabB and PabC.Negative feedback loop is built up upon PABA production as it absorbs UV-B and increases khv.
To decode our light receptor input, we created a full deterministic ODE model. In order to span the ODE system, we employed rule-based models. For this methodology, we defined seed species and rules on how they can interact.
We modeled the system in two ways: with and without negative feedback from PABA and compared the dynamic range.
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ore
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WTGFPTetR +GFPTetR−DBD +GFP
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GFPTetR−DBD +GFPUVR8−TetR−DBD +GFP −IPTGUVR8−TetR−DBD +GFP +IPTGWT
Blue light
Red light
Ptrp
PompC
Ptet_lac
PcI_lac
PcI_tet
cI pabABC Violet
tetR
Green
Red
lacI
Who’s your PABA