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| - | === ''E.Colipse'' project description===
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| - | Sun UV radiation is unavoidable during warm sunny days. Despite the fact that suns light reaching Earth crust consists of only few percent of UV-A (320 - 400 nm) and UV-B (280 - 320 nm) radiation, it still poses serious risks and life destructive capabilities, e.g. DNA is one of the main targets which are hit by UV radiation, causing DNA breakage, mutations etc. (literature) If unattended, this can lead to sun burns, skin damage or even cancer. Thus, a proper skin protection is usually a good idea in order to avoid unpleasant experience.
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| - | However, conventional sunscreens provide relatively passive sun protection, leaving actual protection regulation to ones speculations when and how much of the sunscreen should be applied, which easily can result in inefficient layer of sunscreen and skin damage.
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| - | Thus, we tried to develop an intelligent sunscreen, which could adapt its protection factor to the exposed UV radiation and also give a warning signal if protection is insufficient.
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| - | For this purpose we chose ''E. coli'' as our primer chassis, with a later idea to transfer a working system to a natural bacterial fauna of the skin or alginate beads. As a UV-B light absorbing substance, a Para-Aminobenzoic acid (PABA) was chosen for the following reasons: i) PABA is already commonly used in sunscreens for UV-B protection (literature); ii) PABA is a natural occurring compound as an intermediate in folate synthesis pathway. Furthermore, overproduction of PABA requires overexpression of just two bottleneck enzymes: pabA and pabB, and several attempts were made to overproduce PABA in E.coli by other iGEM teams (literature). Thus we cloned both pabA and pabB into one operon under the constitutive promoter leading to an increase PABA concentration (LET’S HOPE) in the cells. Furthermore, we are not limited to PABA and other natural light absorbance molecules, such as mycosporine-like amino acids, might be used for a final system (literature: review about biosunscreens.
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| - | To couple PABA synthesis to UV light we investigated ''E.coli'' response to UV radiation. However, in prokaryotes such response is activated indirectly by sensing DNA damage (literature). Such protection strategy would be erroneous to pursue, because induction of protection is initiated after DNA damage is already happened. Thus we used plant UV-B responsive protein UVR8 to design a novel UV-B receptor in ''E. coli''.
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| - | In the dark state, UVR8 forms a dimer, which monomerizes upon UV-B irradiation and induces transcriptional changes necessary for plant UV response (literature). However, UVR8 is not able to bind DNA, thus we fused it with a DNA binding domain of tetracycline repressor protein (TetR<sub>DBD</sub>). TetR<sub>DBD</sub> does not contain dimerization domain required for efficient binding and repression of TetR responsive promoter P<sub>tet</sub>. However, external dimerization would in principle restore TetR<sub>DBD</sub> ability to repress P<sub>tet</sub>. Thus, our chimeric protein UVR8-TetR<sub>DBD</sub> acts as an light inducible transcription repressor – at dark state, UVR8 dimer brings TetR<sub>DBD</sub> at close proximity to bind P<sub>tet</sub>, whereas UVR8 monomerization upon UV-B light releases UVR8-TetR<sub>DBD</sub> from DNA, thus inducing transcription of the downstream genes (fig.).
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| - | [[File:UVR8-tetRDBD scheme.png.jpg|frameless|700px|center|thumb|Figure : UVR8-TetR<sub>DBD</sub> working mechanism: in dark state UVR8-TetR<sub>DBD</sub> is a dimer and represses P<sub>tet</sub>, while under exposure of UV-B light UVR8-TetR<sub>DBD</sub> releases DNA and alows transcription of a downstream genes. ]]
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| - | [[Image:NOR_gates.jpg|right|thumb|150px|Figure: NOR gates: Boolean NOR gate representation (upper) and genomic NOR gate - promoter regulated by two repressors (bottom) ]]
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| - | Our models suggest that such system would efficiently induce production of PABA at the right time scale, giving broad possibilities for such novel UV-B switch. Also, our data shows that repression of P<sub>tet</sub> is indeed restored for TetR<sub>DBD</sub>, however, higher inductions of UVR8-TetR<sub>DBD</sub>, required for tighter P<sub>tet</sub> repression, results in the formation of inclusion bodies and cell cytotoxicity. Thus, a second approach of indirect UV sensing was also investigated.
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| - | Each light source has its characteristic light emission spectrum e.g. sun contains high red and blue light continent compared to artificial light sources, such as tungsten light bulb have just high red and a very small fraction of blue light. Thus, we design a biological circuit which can distinguish between four different light conditions: i) high red-high blue; ii) high red-low blue; iii) low red-high blue and iv) low red-low blue (fig.). Such circuit consists of three hybrid promoters each acting as a NOR gate for two different transcriptional repressors i.e. transcription from such promoter is activated just when both repressors are absent.
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| - | We developed a novel photoinduction model to select for a suitable light receptors for the system inputs: LOVtap (blue light receptor) and Cph8 (Red light receptor) (literature). Later analysis of the circuit network revealed several kinetic parameters constrains upon which hybrid promoters were chosen and synthesized. Further testing of the promoters showed that as predicted, they act as a NOR gate, giving high potential of building a first full working light regulated biological decoder circuit in cells.
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| - | [[File:Design.jpg|frameless|700px|center|thumb|Figure : Boolean logic of Decoder. ]]
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