Team:Valencia Biocampus/Results1

From 2012.igem.org

Bacteria Results




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. 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. Legend.

Figure 2. Legend.

Figure 3. Legend.





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 3. Legend.	Figure 4. Legend.





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).

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