Intelligent sun protectionIsaak Müller, Lisa Seyfarth, Gintautas Vainorius, Deborah Huber, Sandro Kundert,

Stefan Ganscha, David Seifert, Tim Enke Advisors: Dr. Johannes Härle, Moritz Lang, Markus Jeschek

Instructors: Prof. Sven Panke, Prof. Jörg Stelling

References: [1] Christie J.M. et al, Science, 355 (2012), 1492., [2] Cox R.S. et al, Molecular systems biology, 3 (2007), 145, [3] Heijde M. et al, Trends in plant science, 17 (2012), 230, [4] Mancinelli A. et al, Plant physiology, 82 (1986), 956, [5] Kinkhabwala, A. et al, PloS one, 3(4) (2008), e2030, [6] Strickland D. et al, PNAS, 105 (2008), 10709

Who’s your PABA?

With the Sun Protection Factor Model we estimated the Sun Protection Factor (SPF) of our PABA producing E. colipse bacteria and vice versa, we could predict the amount of PABA needed to achieve a certain SPF. The SPF, as a rule of thumb, gives you the time in minutes how much longer you can expose yourself to the sun. We learned from the model that PABA alone will not yield a high SPF, since it lacks the capability to absorb dangerous UVA radiation (Fig. 11). The insight here is that another UVA absorbing molecule would drastically increase the sun protection factor of our E. colipse sunscreen.

Protection: PABA, para-Aminobenzoic-acid, is a UV-B absorbing agent that is a common ingredient of sunscreen. It is produced as an intermediate in the folate pathway in a two step reaction from chorismate. We used a chorismate accumulating strain and overproduced PABA by overexpress-ing enzymes pabA and pabBC. The amount of PABA was measured by HPLC (Fig. 10).

Direct UV detection: UVR8

Indirect Sunlight Detection: Decode Sunlight

Project Achievements

Acknowledgements

UV radiation

unprotected sun exposure

protection & warning

signal

The Photoinduction Model allows us to estimate the activity of a light receptor when exposed to a light source. It takes the sources’ light emission spectra and the absorption spectra (Fig. 7), quantum yield and extinction coefficient of the receptor and returns the first order reaction rate kact [s-1] of the receptor for our mechanistical models.[4]

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Fig. 7: Input for Photoinduction model. left: sun photonflux, N calculated from emission spectra; right: light receptor cph-1 absorption spectra to calculate photoconversion cross section

Nλ = photon flux [mol m−2s−1nm−1]σλ = photoconversion cross section [m2mol−1]σλ = 2.303ελΦλ

ελ = molar extinction coefficient [m2mol−1]Φλ = quantum yield, dimensionless, independent of λ

E. colipse is an intelligent and adaptive sun radiation protection system which responds to sunlight 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 of sunlight by engineering a novel UV-B sensitive transcription factor and an indirect detection by incorporating two existing photosensors into a decoder.

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We fused the UV-B light sensing protein UVR8 from Arabidopsis Thaliana[3] to the DNA binding domain of the transcriptional repressor TetR (TetRDBD). Lacking the dimerization domain, TetRDBD is a monomer and thus can not bind to the tet-operator. In its dark state UVR8 forms a dimer which brings TetRDBD in proximity and restoring transcriptional repression. Upon UV exposure the UVR8 domain monomerizes[1] and with it

We used the photoinduction model to decide which light receptors that are available as parts give a good activity under sun light condition and are thus suitable for our indirect light detection with our decoder. Furthermore, it gives us the activation parameter for our mechanistical model of our direct UV - sensing circuit.

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Fig. 1: Overall scheme of direct UV detection. The fusion protein acts as a UV-inducible light switch. UV-B radiation disrupts the UVR8-dimer, forcing the fused TetRDBD todissociate from its operator site.

Fig. 2: Expression of UVR8-TetRDBD. a) SDS PAGE of WT and UVR8-TetRDBD plasmid containing strain. The gel shows that we are able to express our fusion protein in E. coli. b) Native gel of purified fusion protein. Heat treatment leads to monomerization of the UVR8-TetRDBD dimer. Thus we conclude that it is expressed as a dimer.

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Fig. 3: TetRDBD is not able to repress transcription as a monomer, while full length TetR completely represses transcription. Measurement was performed in a platereader.

65 kDa UVR8-TetRDBD

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Fig. 4: UVR8-TetRDBD represses transcriptional output. Results of a platereader assay. The fusion protein was induced with IPTG and thus fluorescence is repressed.

Fig. 11: Absorption spectra of PABA and E. coli themselves. Usujirene, a recently described mycosporine, may be expressed in E. coli and could bridge the gap in absorption between 300 and 400 nm to improve the SPF.

UVR8-TetRDBDWe designed a cross kingdom UV light receptor (BBa_K9090 -07, -08, -09) which is the first engineered UV receptor used in synthetic biology!

Decoder Design, cloning and testing of the hybrid promoters that are at the core of our decoder: They work! (BBa_K9090 -11,-13, -16). Successful cloning of the whole decoder.

PABAWe measured a two-fold increase in produced PABA using our construct (BBa_K9090 -14, -15).

Human practiceWe reviewed how Switzerland deals with genetically modified plants and interviewed two key players, Prof. Ingo Potrykus, founder of the Golden Rice project, and Mr. Markus Ritter.

the TetRDBD, thus enabling transcription (Fig. 1). First, we showed that we are able to express our UVR8-TetRDBD fusion in E. coli (Fig. 2a) and that it occurs as a dimer (Fig. 2b). Second, we showed that TetRDBD alone can not dimerize and repress transcriptional output (Fig. 3). Third, we were able to show that UVR8-TetRDBD is able to repress transcription as a dimer (Fig. 4). Last, we have some evidence that UV radiation induces transcription (Fig. 5) however further investigation is needed. We modeled the complete system (Fig. 6a) with an ODE model to get a grasp on the response time of PABA production and to investigate possible competitive behaviour between UVR8-TetRDBD and PABA for UV-B (Fig. 6b).

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Fig. 6: UVR8-TetRDBD circuit model. a) The system uses UVR8-TetRDBD as a direct UVB sensor. The negative feedback, indicated by the dotted line, comes from the fact that PABA absorbs UV while UV activates our fusion protein and subsequently the production of PABA. b) Modeling revealed that the time scale of PABA production is around an hour after sunlight exposure. Furthermore, the negative feedback did lower the amount of PABA produced. Sunlight exposure is at 4 h.

Like a parasol that protects indirectly from the sun the decoder senses sunlight as the presence of both red and blue light. It distinguishes sunlight from other light sources such as a bulb (emits mostly red light) or a fluorescent lamp (emits mostly blue light) (Fig. 8). It detects sunlight via its unique composition of both blue and red light. The design is based on logic circuits: We use biological NOR gates wired together to form our 2-to-4 decoder system. For the biological implementation, we designed and tested double repressive hybrid promoters[2],[5] (Fig.9) based on the operator sites of the transcriptional repressors TetR, LacI and CI. Before implementing the system, we modeled the decoder with an ODE model. Optimization of the separation of the decoder outputs gave us some constraints on the promoter strengths. Using FACS we showed that two of our hybrid promoters work as expected. The whole decoder was successfully cloned into E. coli. Cells on agar plates show, as expected, a mixed signal and lower fluorescence intensity. This serves as a first insight that the system might work as a whole.

Fig. 10: HPLC measurement to detect PABA. From left to right: PABA molecule, HPLC results for our pabA-pabBC construct, for the wildtype and for pure industrially synthesized PABA (Sigma-Aldrich). By calculating the integral of the peak, which is directly proportional to the amount of PABA in the sample, we could deduce that our construct yields twice the amount of PABA compared to the wildtype.

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Fig. 9: a) FACS of two hybrid promoters. We cotransfromed two of our hybrid promoters with a second plasmid for constitutive expression of LacI and TetR. An overnight culture was tested using four conditions: Induction with IPTG, with aTc, with both and without induction. FACS data shows four distinct subpopulations. Additionally we deduce that mCherry expression is stronger than eCFP due to different promoter strength.b) Agar plates with transformed E. coli from a colony PCR. Even with inspection by eye the reporters under the hybrid promoters can be seen. Top: mCherry under control of CI and TetR repressible promoter. Lower left: eCFP under control of CI and LacI repressible promoter. Lower right: Our whole transformed decoder. Here CI represses mCherry and eCFP production, therefore the color is dimmer. Note that on these plates the plasmid containing the repressors LacI and TetR was not cotransformed.

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Jeff Tabor, Rice Universitz (Houston), for providing the JT2-strain and the pJT122 and pJT118 plasmidsPeter Kast, ETH Zurich, for providing the (chorismate overproducing) E. coli strain K12Roman Ulm, University of Geneva, for providing the cDNA of UVR8 from Arabidopsis thaliana

We thank Team Uppsala 2012 for providing their low-copy backbones and chromoproteins

Daniel Gerngross, responsible for logo design, groupmember pictures and the team picture

Our Mamas and PABAs

From BSSE, Panke and Stelling group:Sonja Billerbeck for theoretical and practical helpAndreas Bosshart for providing plasmids for testingGaspar Morgado for advice considering operator sitesChristian Mayer for providing his software Ru2renFabian Rudolf for discussions on UVR8-TetRDBDVerena Jaeggin for her support using FACS

We want to specially thank our Advisors who were available for our constant questions throughout the whole project: Johannes Haerle, Markus Jeschek and Moritz Lang.

colipseESwitzerland

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Fig. 5: UVR8-TetRDBD is no more able to repress transcription when exposed to UVB. FACS data indicates through a shift in the fluorescent maximum, indicated with the arrows, that transcription is restored when UVR8-TetRDBD is exposed to UV.

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sunlight 1 1 0 0 1 eYFP

Fig. 8: Overall scheme of the biological implementation of the decoder and logic table of input, internal states and output. Note that we can simulate red light with aTc and blue light with IPTG to easily test our system. The hybrid promoters serve as biological NOR gates: if one of the transcriptional regulators is present, the output is repressed. Three NOR gates arranged like this form a 2-to-4 decoder: If no light is present there is no fluorescent output. One wavelength alone induces a respective reporter gene, ecfp or mcherry, but it cannot trigger the main output in the middle, here shown with the reporter eyfp. Only sunlight, which contains both red and blue lightwaves, induces eYFP production, while additionally producing CI to repress both other reporters.

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