Team:Heidelberg LSL/Measurement

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Measurement

Background

For realizing our vision to construct bacteria that detect UV and radioactive radiation and give a color output visible by eye, we constructed three different sensor parts based on the E. coli SOS-response promoters. These are the pomoters pRecA, pRecB and pSulA combined with a LacZ reporter. E. coli bacteria transformed with our sensors show a highly sensitive, UV-dose dependet coloring, as measured by both ONPG (figure 1) and X-Gal assays (figure 2). We show that our promoters also work in combination with other reporters such as GFP (figure 3), proofing the modularity of the approach we propose. Finally, we proof the performence of two submitted parts (pRecA-LacZ, part BBa_K862000 and pSulA-LacZ, part BBa_K862001) in an outdoor experiment, where we really induced our bacteria in the summer sun (figure 4). Taken together, we show that our sandardized DNA-damage sensors provide an easy and highly sensitive approach for the detection of UV radiation, which can in principle be extended to other radiation sources, such as radioactive radiation and could even be used for the detection of DNA damaging agencies in the future.

Measurements Principle – our Part Characterization

We transformed our UV radiation measurement constructs, namely parts BBa_K862000, BBa_K862001 and BBa_K862002 into BL21(DE3) bacteria (recA+ strain). The basic measurement strategy we used was illumination of bactrial o/n cultures in 6- or 24- well plate formats in a UV geldoc chamber for different time periods. We primarlily used beta galactosidase (LacZ) as a reporter and added to substrate (ONPG giving a yellow color with a max. absorbance at 420 nm or X-Gal giving a blue color with a max. absorbance at 610 nm) after illumination of the samles.Color development was quantified using a plate reader (ONPG assay) or by calculating the color intensities from RGB pictures taken with a digital camera using ImageJ (X-Gal assay). For our GFP reporter test, bright-field fluorescence microscopy was applied and GFP expression was quantified from pictures taken from UV-irradiated verus non-irradiated bacteria.

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 <h4>Background</h4>
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-For realizing our vision to construct bacteria that detect UV and radioactive radiation and give a color output visible by eye, we <a href="https://2012hs.igem.org/Team:Heidelberg_LSL/Project_UVsensors">constructed three different sensor parts</a> based on the E. coli <a href="https://2012hs.igem.org/Team:Heidelberg_LSL/Project_SOS">SOS-response promoters</a>. These are the pomoters pRecA, pRecB and pSulA combined with a LacZ reporter. E. coli bacteria transformed with our sensors show a highly sensitive, UV-dose dependet coloring, as measured by both ONPG (figure 1) and X-Gal assays (figure 2). We show that our promoters also work in combination with other reporters such as GFP (figure 3), proofing the modularity of the approach we propose.
+For realizing our vision to construct bacteria that detect UV and radioactive radiation and give a color output visible by eye, we <a href="https://2012hs.igem.org/Team:Heidelberg_LSL/Project_UVsensors">constructed three different sensor parts</a> based on the <i>E. coli</i> <a href="https://2012hs.igem.org/Team:Heidelberg_LSL/Project_SOS">SOS-response promoters</a>. These are the pomoters pRecA, pRecB and pSulA combined with a LacZ reporter. <i>E. coli</i> bacteria transformed with our sensors show a highly sensitive, UV-dose dependet coloring, as measured by both ONPG (figure 1) and X-Gal assays (figure 2). We show that our promoters also work in combination with other reporters such as GFP (figure 3), proofing the modularity of the approach we propose.
 Finally, we proof the performence of two submitted parts (pRecA-LacZ, part BBa_K862000 and pSulA-LacZ, part BBa_K862001) in an outdoor experiment, where we really induced our bacteria in the summer sun (figure 4).
 Taken together, we show that our sandardized DNA-damage sensors provide an easy and highly sensitive approach for the detection of UV radiation, which can in principle be extended to other radiation sources, such as radioactive radiation and could even be used for the detection of DNA damaging agencies in the future.