Team:ETH Zurich/Project

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== Overview ==  
== Overview ==  
===  [https://2012.igem.org/Team:ETH_Zurich/UVR8 UVR8-TetR<sub>DBD</sub>] ===
===  [https://2012.igem.org/Team:ETH_Zurich/UVR8 UVR8-TetR<sub>DBD</sub>] ===
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[[File:UVR8.png|frameless|350px|right|thumb|Binding of TetR to the ''tetO'' <span class='eth_reference'>[Orth2000]</span>]]
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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 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, a UV-B-detecting-protein from plants, in different ways  - repression occured.
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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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===  [https://2012.igem.org/Team:ETH_Zurich/LovTAP LovTAP] ===
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<br><br><br><br><br><br><br>
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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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===[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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===  [https://2012.igem.org/Team:ETH_Zurich/Cph8 Cph8] ===
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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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<br style="clear: both" />
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=== [https://2012.igem.org/Team:ETH_Zurich/Decoder Decoder] ===
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[[File:Design.jpg|frameless|500px|right|thumb|Boolean logic of Decoder]]
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Our decoder aims the combination of blue and red light to distinguish 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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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

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  • 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.
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  • 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.
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  • 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.
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