Team:Valencia Biocampus/Results1

Are you hungry?

The experiments with our glucose-sensitive construction were carried out by three different ways: (1) containing only glucose as a carbon source, (2) containing galactose as an extra carbon source and (3) containing sodium acetate as an extra carbon source, since we checked in previous tests that low concentrations of glucose compromised cell growth. All tubes had, in addition to the glucose gradation, also IPTG (see information on the molecular mechanism). For fluorescence intensity (FI) measures cell growth, i.e. OD600, was taken into account. We worked with a 0D600 close to 0.1 in order to be able to use high sensitivity in the fluorimeter.

As you can see in the graphs below, the less glucose you have, the more fluorescence you get. And this results replicate well when galactose (Figure 2) or sodium acetate (Figure 3) were added to the medium as supplementary carbon sources. The threshold for a boost in the fluorescence intensity seems to be 0.1 g/L of glucose, since from this concentration the values get really high. Normal values are 10 g/L, and we got the best results when we added 10^-4 glucose concentration of that of the canonical values.

Figure 1. Fluorescence intensity (FI) normalized by the optical density of the culture (OD) for differents amounts of glucose present in the medium.

Figure 2. Fluorescence intensity (FI) normalized by the optical density of the culture (OD) for differents amounts of glucose in a medium supplemented with 3 g/L galactose.

Figure 3. Fluorescence intensity (FI) normalized by the optical density of the culture (OD) for differents amounts of glucose in a medium supplemented with 3 g/L sodium acetate.

Do you have enough nitrogen

In order to characterize our nitrogen-sensitive construction, we carried out an experiment as follows: our E.coli strain carrying the ZsYellow1 gene under the control of the glnA promoter was grown on LBA medium (until an OD of 1.5) in order the bacteria to have sufficient amounts of proteins and nitrogen compounds. Then, cells were pelleted and resuspended in a medium lacking nitrogen. After that, values of OD and fluorescence intensity were measured at different times. As a control, we resuspended one of the aliquots in medium containing 10 g/L of ammonium sulphate.

As expected according to the underlying molecular mechanism, nitrogen starvation induces the expression of the fluorescent protein:

Figure 4. Fluorescence intensity (FI) normalized by the optical density (OD) of the culture. Measures were taken 60 min after the resuspension in a medium containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).		Figure 5. Fluorescence intensity (FI) normalized by the optical density (OD) of the culture. Measures were taken 150 min after the resuspension in a medium containing no nitrogen (pink) or 10 g/L ammonium sulphate (grey).

Can you breathe?

In this experiment, our E.coli strain carrying the ZsGreen1 protein under the control of the nirB promoter was grown on LBA under two different conditions: aerobic and anaerobic. Anaerobic conditions were achieved by adding a small volume of sterile oil over the liquid culture (Figure 6), so no oxygen can be taken from the environment. After 2 days of growth, samples were taken from each culture and diluted to an OD of 0.1. After that, fluorescence intensity was measured.

Our results show that ZsGreen1 expression increases (a 20% approx.) under anaerobic conditions, according to the effect of oxygen on the FNR proteins (see the molecular mechanism description).

Figure 6. Fluorescence intensity (FI) normalized by the optical density (OD) of cultures grown under aerobic (grey) or anaerobic (pink) conditions.

Are you hot?

To test the heat-sensitive construction, an overnight culture of our E.coli strain carrying the AsRed2 gene under the control of groE promoter was set up at 25 ºC. After that, OD was adjusted to 0.15 and several aliquots were transferred to fluorimetry cuvettes. Heat shock was carried out at different temperatures in a water bath. Then, the cuvettes were maintained at room temperature and OD and fluorescence intensity were measured at different time points.

During the first experiments, we did not get any successful result... As we can see in the following graph, fluorescence emission showed no significant increasement after different heat-shock treatments:

Figure 7. Fluorescence intensity (FI) normalized by the optical density (OD) of cultures subjected to a 44 ºC heat-shock treatment of different durations. Heat-shock was not applied in the control experiment.

That was suspicious! Since we were sure that our experimental set-up was fine, we decided to check if our bacteria were actually carrying the right construction (Bac2, that consisted of groE promoter + AsRed2). Then, we found out that we had lying bacteria in the lab! Cheaters!

After that, we started to perform new experiments with "honest" bacteria, and good results arrived soon: as shown in the graphs below, we found a correlation between the temperature at which heat shock was carried out and the level of expression of the fluorescent protein. Higher fluorescence intensity was obtained when higher temperatures (ranging from 40 to 44 ºC) were applied (check the molecular mechanism details). According to our results, it seems that protein expression cannot keep increasing at temperatures higher than 44 ºC, probably due to lethality effects.

Figure 8. Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that were subjected to a 10-min heat-shock at different temperatures. Measures were taken 60 min after the shock.

Figure 9. Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that were subjected to a 10-min heat-shock at different temperatures. Measures were taken 120 min after the shock.

Express your gene!

In order to test our UV-induced construction, the E. coli strain carrying the ZsGreen1 gene under the control of the lexA promoter was grown on LBA (LB broth supplemented with ampicillin) until an OD value of 0.8. Then, cells were pelleted, resuspended in fresh medium, and 100 uL were spread in agar plates. Once the medium was completely absorbed by the agar (to avoid UV absorption by medium), cells were irradiated inside the TORDEITOR (the device we specially designed for this experiment) with different amounts of UV light (lambda=254 nm). To fine tune UV dose, we used a shutter (similar to those used in cameras, click here to see an explicative video) able to produce UV pulses of less than 1 second of duration. After that, cells were harvested from the agar plate with SOC medium supplemented with ampicillin. Finally, OD and fluorescence intensity were measured at different times. An aliquot of the same culture that was not irradiated was used as control.

As we can see in the graph below, UV exposure does induce the expression of ZsGreen1. Furthermore, longer exposures result in higher levels of protein expression (higher fluorescence intensity was observed, as expected according to the molecular mechanism of lexA promoter), although lethal effects were observed when doses about 5 seconds were applied.

Figure 10. Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that were subjected to UV exposures of different duration in comparison to a control culture (not exposed). Measures were taken 70 min after the exposure.		Figure 11. Fluorescence intensity (FI) normalized by the optical density (OD) of cultures that were subjected to UV exposures of different duration in comparison to a control culture (not exposed). Measures were taken 90 min after the exposure.