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

0 2 4 6 80

0.5

1

1.5

2x 104

Time / h

Con

cent

ratio

n / n

M

0 2 4 6 80

2000

4000

6000

8000

10000

Time / h

Con

cent

ratio

n / n

M

0 2 4 6 80

200

400

600

800

Time / h

Con

cent

ratio

n / n

M

PabAB

0 2 4 6 80

1000

2000

3000

4000

5000

6000

7000

Time / h

Con

cent

ratio

n / n

M

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

200 300 400 500 600 700 800 900 10000

0.5

1

1.5Irradiance at probe

Irrad

ianc

e [W

m−2

]

λ [nm]

sun

200 300 400 500 600 700 800 900 10000

1

2

3

4

5

6

7

8x 10−6 Photon flux

Irrad

ianc

e [m

ol m

−2]

λ [nm]

sun

200 300 400 500 600 700 800 900 10000

100

200

300

400

500

600

700Photoconversion

rel.

phot

ocon

vers

ion

[m2 m

ol−1

]

λ [nm]

lov−a

200 300 400 500 600 700 800 900 10000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1Absorption spectrum lov−a

A.U

.

λ [nm]

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

0 2 4 6 80

5

10

15

Con

cent

ratio

n / n

M

Green

0 2 4 6 80

5

10

15Red

0 2 4 6 80

5

10

15Violet

0 2 4 6 80

5

10

15

Con

cent

ratio

n / n

M

0 2 4 6 80

5

10

15

0 2 4 6 80

5

10

15

0 2 4 6 80

5

10

15

Con

cent

ratio

n / n

M

0 2 4 6 80

5

10

15

0 2 4 6 80

5

10

15

0 2 4 6 80

5

10

15

Time / h

Con

cent

ratio

n / n

M

0 2 4 6 80

5

10

15

Time / h0 2 4 6 8

0

5

10

15

Time / h

RedBlue

RedBlue

RedBlue

RedBlue

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.

0 2 4 6 8 10

05000

10000

15000

Time[h]

Flu

ore

scence

/OD

600

WTGFPTetR +GFPTetR−DBD +GFP

0 2 4 6 8 10

050

0010

000

1500

0

Time[h]

Flu

ores

cenc

e/O

D60

0

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