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.

1) ONPG assay for the sensitive measurement of reporter activity already after very short UV exposure times

In order to determine the response of our SulA and RecA promoters to low UV doses, we used the highly sensitive ONPG assay.
ONPG (Ortho-nitrophenyl-β-D-galactopyranoside) is a synthetic LacZ substrate and produces a yellow color (o-nitrophenol) upon cleavage by LacZ. The formation of the yellow color can be easily determined using a photometer at 420 nm absorbance wavelength. 0.5 ml of the overnight culture of Bl21(DE3) transformed with our RecA_LacZ or SulA_LacZ constructs were put into each well of a 6-well plate. Cells were irradiated in a Geldoc-UV-Chamber for 0-600 s. 10 min after irradiation, an ONPG-Assay was performed in a 96-well format (using technical duplicates).


Fig. 1: ONPG assay of Bl21(DE3) irradiated for different times. Both constructs show a strong correlation between the UV-irradiation time and the LacZ activity (production of o-nitrophenol).

Both, the SulA_LacZ and the RecA_LacZ constructs, show a nice increase of LacZ activity with increasing UV irradiation times already 10 min after induction. This argues for a rapid response by our sensor constructs which is due to the overall rapid SOS response initiated by the UV radiation. The OD did not change with UV radiation, showing that the cell number was not effected by the UV treatment.

2) X-Gal Assay for the characterization of our parts in an application-relevant context

For chracterizing the RecA,B and SulA constructs with lacZ reporter in a close-to application context, we performed X-Gal assays. Therefore, we put o/n cultures of E. coli transformed with our sensor parts onto 24 well plates (12 of the same culture samples, 0.6 ml/well, each construct onto a seperate plate). We induced the plates for 0 s and transfered the first 2 samples onto a several plate. After 5 min induction we put the second two samples onto a serperate plate and so on. Thereby, we subsequently induced our samples and got induction times between 0 and 30 min with 2 replicates for each induction time. The start point of induction is the same for all samples. After 1 h incubation at 37 °C/80 rpm we added X-Gal substrate (final concentration of 200 µg/ml) to all samples. Coloring of the wells were monitored by taking pictures 15 min after X-gal addiation (figure 2, left). Quantifications of the coloring intensity were done from the pictures taken by pixel grey-value analysis in ImageJ in the different wells (figure 2, right).


Fig. 2: X-gal assay of Bl21(DE3) transformed with RecA/RecB/SulA_LacZ parts and irradiated for different times. All constructs show a strong positive correlation between UV induction time and coloring of the wells. RecA-LacZ gives the lowest reporter background expression wheras SulA-LacZ gives the highes overall coloring of the samples.

All constructs show a clear, UV dose-dependent coloring, that is visible already by eye, showing that the constructs are working really great. Whereas the RecA construct has the lowest promoter background activity (almost no coloring at 0 min timepoint), the SulA shows the largest color development for high UV doses (20 and 30 min). Therefore, all promoters have slightly different properties and would have certain advantages or disadvantages in different appication contexts (i.e. in short or long-term UV measurements).

3) Test of alternative reporter: GFP fluorescence induction using our RecA-GFP construct

For investigating, whether our approach is also working for other reporters than lacZ, we tested our RecA-GFP construct under similar conditions. An overnight culture of E. coli BL21(DE3) transformed with RecA-GFP was distributed onto 2 6-well plates (3 ml/well, experiment done in duplicates) and either induced by UV-irradiation for 30 min or left uninduced. 30 min after induction fluorescence microscopy was performed (excitation at 470 nm, GFP emission filter). GFP-Expression was quantified from the microscope pictures using ImageJ.


Fig. 3: Test of RecA-GFP reporter construct by fluorescence microscopy. E. coli transformed with our RecA-GFP reporter were either UV-irradiated for 30 min or left uninduced. measurement of GFP expression show a 10-fold (!) increase in GFP expression due to UV-irradiation compared to the control.

E. coli transformed with our RecA-GFP measurement constructs show a 10 fold (!) induction of GFP expression after 30 min of UV irradiation compared to the non-irradiated control. This proves that our modular approach of combining endogenous SOS-promoters such as RecA, RecB and SulA with different reporters works really fine. Therefor, we give users of our constructs the opportunity to measure radiation by using different measurement setups (X-gal assay, fluorescence microscopy and potentially many more).