Team:ETH Zurich/Project

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== Overview ==  
== Overview ==  
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In this section we show the experiment achievement.
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===  [https://2012.igem.org/Team:ETH_Zurich/UVR8 UVR8-TetR<sub>DBD</sub>] ===
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[[File:UVR8.png|frameless|250px|right|thumb|Fig. 1 UVR8 homodimer <span class='eth_reference'>[Christie2012]</span>]]
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We introduce and implement a novel two-hybrid screening for ''E.coli'' using the TetR-DNA binding domain. TetR<sub>DBD</sub> on its own is unable to bind to the P<sub>tet</sub> promoter. After fusing it to the dimerizing UVR8 (Fig. 1), a UV-B-detecting-protein from plants, in different ways  - repression occured.
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===  [https://2012.igem.org/Team:ETH_Zurich/Decoder Decoder] ===
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Our decoder (Fig. 2) aims to combine blue and red light to distinguish between different lightsources like sunlight or room light. For this we use LovTAP and Cph8 and perform the calculation with the help of three NOR gates each represented by a new designed hybrid promoter.
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LovTAP is a fusion of a photoreceptor with a transcriptional regulator TrpR. This fusion protein can be activated by blue light and therefore act as a transcriptional switch. We want to use this system for the light intensity measurement and as an input for our decoder.
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Cph8 consists of the light-receptor Cph1 that is deactivated by red light and the histidine kinase EnvZ responsible for the downstream signaling. We aim to use it for our decoder as a red-light-sensor.
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[[File:Design.jpg|frameless|500px|left|thumb|Fig. 2 Boolean logic of Decoder]]
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=== UVR8-TetR<sub>DBD</sub> ===
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<br><br><br><br><br><br><br>
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We implement a novel two-hybrid screening for E.Coli using the TetR-DNA binding domain. TetR<sub>DBD</sub> on its own is unable to bind to the Tet-Promoter. After fusing it in different ways to UVR8 - a UV-B-detecting-protein from plants - repression occured.
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===[https://2012.igem.org/Team:ETH_Zurich/PABA Para-Aminobenzoic acid]===
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[[Image:PABA.png|left|thumb|100px|''Figure 2'': 4-para-aminobenzoic acid, natural occuring UV light absorbing molecule used in sunscreens]]
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As an output of either our direct or our indirect sun detection we are planing to produce para-Aminobenzoic acid (PABA). PABA is able to absorb UVB light and therefore will act as an active ingredient in our intelligent sunscreen.
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In paralel to PABA we are going to produce a violet pigment. This will serve as a warning signal to avoid further sun exposure.
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Latest revision as of 23:31, 26 October 2012

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Contents

Overview

UVR8-TetRDBD

Fig. 1 UVR8 homodimer [Christie2012]

We introduce and implement a novel two-hybrid screening for E.coli using the TetR-DNA binding domain. TetRDBD on its own is unable to bind to the Ptet promoter. After fusing it to the dimerizing UVR8 (Fig. 1), a UV-B-detecting-protein from plants, in different ways - repression occured.

Decoder

Our decoder (Fig. 2) aims to combine blue and red light to distinguish between different lightsources like sunlight or room light. For this we use LovTAP and Cph8 and perform the calculation with the help of three NOR gates each represented by a new designed hybrid promoter. LovTAP is a fusion of a photoreceptor with a transcriptional regulator TrpR. This fusion protein can be activated by blue light and therefore act as a transcriptional switch. We want to use this system for the light intensity measurement and as an input for our decoder. Cph8 consists of the light-receptor Cph1 that is deactivated by red light and the histidine kinase EnvZ responsible for the downstream signaling. We aim to use it for our decoder as a red-light-sensor.

Fig. 2 Boolean logic of Decoder









Para-Aminobenzoic acid

Figure 2: 4-para-aminobenzoic acid, natural occuring UV light absorbing molecule used in sunscreens

As an output of either our direct or our indirect sun detection we are planing to produce para-Aminobenzoic acid (PABA). PABA is able to absorb UVB light and therefore will act as an active ingredient in our intelligent sunscreen.

In paralel to PABA we are going to produce a violet pigment. This will serve as a warning signal to avoid further sun exposure.


References

  • Brown, B. a, Headland, L. R., & Jenkins, G. I. (2009). UV-B action spectrum for UVR8-mediated HY5 transcript accumulation in Arabidopsis. Photochemistry and photobiology, 85(5), 1147–55.
  • Christie, J. M., Salomon, M., Nozue, K., Wada, M., & Briggs, W. R. (1999): LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proceedings of the National Academy of Sciences of the United States of America, 96(15), 8779–83.
  • Christie, J. M., Arvai, A. S., Baxter, K. J., Heilmann, M., Pratt, A. J., O’Hara, A., Kelly, S. M., et al. (2012). Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science (New York, N.Y.), 335(6075), 1492–6.
  • Cloix, C., & Jenkins, G. I. (2008). Interaction of the Arabidopsis UV-B-specific signaling component UVR8 with chromatin. Molecular plant, 1(1), 118–28.
  • Cox, R. S., Surette, M. G., & Elowitz, M. B. (2007). Programming gene expression with combinatorial promoters. Molecular systems biology, 3(145), 145. doi:10.1038/msb4100187
  • Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauss, U., Guterl, J.-K., Wendorff, M., et al. (2007). Reporter proteins for in vivo fluorescence without oxygen. Nature biotechnology, 25(4), 443–5
  • Drepper, T., Krauss, U., & Berstenhorst, S. M. zu. (2011). Lights on and action! Controlling microbial gene expression by light. Applied microbiology, 23–40.
  • EuropeanCommission (2006). SCIENTIFIC COMMITTEE ON CONSUMER PRODUCTS SCCP Opinion on Biological effects of ultraviolet radiation relevant to health with particular reference to sunbeds for cosmetic purposes.
  • Elvidge, C. D., Keith, D. M., Tuttle, B. T., & Baugh, K. E. (2010). Spectral identification of lighting type and character. Sensors (Basel, Switzerland), 10(4), 3961–88.
  • GarciaOjalvo, J., Elowitz, M. B., & Strogatz, S. H. (2004). Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proceedings of the National Academy of Sciences of the United States of America, 101(30), 10955–60.
  • Gao Q, Garcia-Pichel F. (2011). Microbial ultraviolet sunscreens. Nat Rev Microbiol. 9(11):791-802.
  • Goosen N, Moolenaar GF. (2008) Repair of UV damage in bacteria. DNA Repair (Amst).7(3):353-79.
  • Heijde, M., & Ulm, R. (2012). UV-B photoreceptor-mediated signalling in plants. Trends in plant science, 17(4), 230–7.
  • Hirose, Y., Narikawa, R., Katayama, M., & Ikeuchi, M. (2010). Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter. Proceedings of the National Academy of Sciences of the United States of America, 107(19), 8854–9.
  • Hirose, Y., Shimada, T., Narikawa, R., Katayama, M., & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences of the United States of America, 105(28), 9528–33.
  • Kast, Asif-Ullah & Hilvert (1996) Tetrahedron Lett. 37, 2691 - 2694., Kast, Asif-Ullah, Jiang & Hilvert (1996) Proc. Natl. Acad. Sci. USA 93, 5043 - 5048
  • Kiefer, J., Ebel, N., Schlücker, E., & Leipertz, A. (2010). Characterization of Escherichia coli suspensions using UV/Vis/NIR absorption spectroscopy. Analytical Methods, 9660. doi:10.1039/b9ay00185a
  • Kinkhabwala, A., & Guet, C. C. (2008). Uncovering cis regulatory codes using synthetic promoter shuffling. PloS one, 3(4), e2030.
  • Krebs in Deutschland 2005/2006. Häufigkeiten und Trends. 7. Auflage, 2010, Robert Koch-Institut (Hrsg) und die Gesellschaft der epidemiologischen Krebsregister in Deutschland e. V. (Hrsg). Berlin.
  • Lamparter, T., Michael, N., Mittmann, F., & Esteban, B. (2002). Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site. Proceedings of the National Academy of Sciences of the United States of America, 99(18), 11628–33.
  • Levskaya, A. et al (2005). Engineering Escherichia coli to see light. Nature, 438(7067), 442.
  • Mancinelli, A. (1986). Comparison of spectral properties of phytochromes from different preparations. Plant physiology, 82(4), 956–61.
  • Nakasone, Y., Ono, T., Ishii, A., Masuda, S., & Terazima, M. (2007). Transient dimerization and conformational change of a BLUF protein: YcgF. Journal of the American Chemical Society, 129(22), 7028–35.
  • Orth, P., & Schnappinger, D. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature structural biology, 215–219.
  • Parkin, D.M., et al., Global cancer statistics, 2002. CA: a cancer journal for clinicians, 2005. 55(2): p. 74-108.
  • Rajagopal, S., Key, J. M., Purcell, E. B., Boerema, D. J., & Moffat, K. (2004). Purification and initial characterization of a putative blue light-regulated phosphodiesterase from Escherichia coli. Photochemistry and photobiology, 80(3), 542–7.
  • Rizzini, L., Favory, J.-J., Cloix, C., Faggionato, D., O’Hara, A., Kaiserli, E., Baumeister, R., et al. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science (New York, N.Y.), 332(6025), 103–6.
  • Roux, B., & Walsh, C. T. (1992). p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA. Biochemistry, 31(30), 6904–10.
  • Strickland, D. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences of the United States of America, 105(31), 10709–10714.
  • Sinha RP, Häder DP. UV-induced DNA damage and repair: a review. Photochem Photobiol Sci. (2002). 1(4):225-36
  • Sambandan DR, Ratner D. (2011). Sunscreens: an overview and update. J Am Acad Dermatol. 2011 Apr;64(4):748-58.
  • Tabor, J. J., Levskaya, A., & Voigt, C. A. (2011). Multichromatic Control of Gene Expression in Escherichia coli. Journal of Molecular Biology, 405(2), 315–324.
  • Thibodeaux, G., & Cowmeadow, R. (2009). A tetracycline repressor-based mammalian two-hybrid system to detect protein–protein interactions in vivo. Analytical biochemistry, 386(1), 129–131.
  • Tschowri, N., & Busse, S. (2009). The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes & development, 522–534.
  • Tschowri, N., Lindenberg, S., & Hengge, R. (2012). Molecular function and potential evolution of the biofilm-modulating blue light-signalling pathway of Escherichia coli. Molecular microbiology.
  • Tyagi, A. (2009). Photodynamics of a flavin based blue-light regulated phosphodiesterase protein and its photoreceptor BLUF domain.
  • Vainio, H. & Bianchini, F. (2001). IARC Handbooks of Cancer Prevention: Volume 5: Sunscreens. Oxford University Press, USA
  • Quinlivan, Eoin P & Roje, Sanja & Basset, Gilles & Shachar-Hill, Yair & Gregory, Jesse F & Hanson, Andrew D. (2003). The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol. The Journal of biological chemistry, 278.
  • van Thor, J. J., Borucki, B., Crielaard, W., Otto, H., Lamparter, T., Hughes, J., Hellingwerf, K. J., et al. (2001). Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1. Biochemistry, 40(38), 11460–71.
  • Wegkamp A, van Oorschot W, de Vos WM, Smid EJ. (2007 )Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis. Appl Environ Microbiol. Apr;73(8):2673-81.