Team:Heidelberg LSL/Measurement

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

For chracterizing the precA, precB and psulA constructs with lacZ reporter (partsregistry: BBa_K862000, BBa_K862002 and BBa_K862001)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 precA/precB/psulA_LacZ parts and irradiated for different times. All constructs show a strong positive correlation between UV induction time and coloring of the wells. PrecA-LacZ gives the lowest reporter background expression whereas psulA-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 precA construct has the lowest promoter background activity (almost no coloring at 0 min timepoint), the psulA 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 precA-GFP construct

For investigating, whether our approach is also working for other reporters than lacZ, we tested our precA-GFP construct under similar conditions. An overnight culture of E. coli BL21(DE3) transformed with precA-GFP (see construct #1 in the sensor construction page 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.

4) Outdoor-Test und real-life application conditions

Finally, in order to test our UV radiation sensor construct in a real application setting, we went outside and used the rarely shining German sun as UV irradiation device. First, o/n cultures with E. coli transformed with either precA-LacZ or psulA-LacZ were put into two different 6-well plates (3 ml/well). The 6-well plates were tightly sealed, glued and additionly wrapped with parafilm. Afterwards, the plate covers were thoroughly desinfected using 70 % ethanol and taken outside. The plates were placed either in the bright sun (it was an average early-summer day) or in the shade. Afterwards, the plates were taken back to the lab and incubated for 30 min at room temperature in the dark. Finally, 2 ml of each of the 4 samples (precA_LacZ and psulA_LacZ, each either incubated in the sun or in the shadow) were pipetted into a cuvette and X-gal was added to a final concentration of 200 µg/ml. Then pictures were subsequently taken every minute while the blue color was developing (we started taking the pictures 2 min after X-gal addition).


Fig. 4: real application outdoor test of precA_LacZ and psulA_LacZ in the summer sun. In order to make a final evaluation of our UV radiation sensor approach, we incubated bacteria transformed with our precA_LacZ and psulA_LacZ in the summer sun for 70 min or in the shade as negative control. Afterwards, we put our bacteria into cuvettes, added X-gal and monitored the development of the blue color over time. Obviously, both constructs allowed a clear distinction between the bacteria incubated in the sun and in the shade. The precA_LacZ construct performed better than the psulA_LacZ, as the precA_LacZ-Promoter had almost no background activity when bacteria were incubated in the shade.

Wrap-up

Our analysis clearly proof that our radiation sensor constructs are highly sensitive, even to low UV irradiation doses (see fig. 1, ONPG-assay), show a clear, broad UV-dose dependent activation dynamics (see fig. 2, X-gal assay) and are even working in a real application setup (see fig. 4, outdoor test). By using a LacZ reporter gene, we enable a very simple measurement approach, where to user can simply monitor the blue color development by eye without the need for any complicated or expensive measurement equipment.
In addition, the approach we propose here is highly modular, so that our promoters, namely precA, precB and psulA could be combined with all different kinds of reporters, which we exemplified by using both LacZ and GFP as reporter (see fig. 3, precA-GFP test).