Team:Heidelberg LSL/Measurement

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<p>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 <i>E. coli</i> BL21(DE3) transformed with precA-GFP (see construct #1 in <a href= "http://2012hs.igem.org/Team:Heidelberg_LSL/Project_UVsensors" > the sensor construction page </a> 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.<br/>
<p>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 <i>E. coli</i> BL21(DE3) transformed with precA-GFP (see construct #1 in <a href= "http://2012hs.igem.org/Team:Heidelberg_LSL/Project_UVsensors" > the sensor construction page </a> 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.<br/>
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<b>Fig. 3: Test of precA-GFP reporter construct by fluorescence microscopy. </b><i>E.coli</i> transformed with our precA-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. <br/>
<b>Fig. 3: Test of precA-GFP reporter construct by fluorescence microscopy. </b><i>E.coli</i> transformed with our precA-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. <br/>

Revision as of 10:40, 18 July 2012

iGEM-2012HS - LSL-Heidelberg iGEM-2012HS - LSL-Heidelberg

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 promoters precA, precB and psulA combined with a LacZ reporter. E.coli bacteria transformed with our sensors show a highly sensitive, UV-dose dependent 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 standardized 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 primarily 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 samples. 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 versus 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 precA_lacZ or psulA_acZ 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 psulA_LacZ and the precA_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 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 separate 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 serparate 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 addition (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 highest 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 time point), 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 application 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.

Fig. 3: Test of precA-GFP reporter construct by fluorescence microscopy. E.coli transformed with our precA-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 precA-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 precA, precB and psulA 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).

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 additionally 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).