Part:BBa_K3463019:Experience

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Characterizationof BBa_K3463019

The purpose of this experiment was to evaluate the correlation between the BHL concentration in the medium and the intensity of the fluorescence emitted.

To evaluate if our system works, we made 6 bacterial cultures of transformed E.coli Nissle with a gradient of synthetic BHL in the medium. Once all the media have been set up at T0 with 100µM / 10µM / 1µM / 100nM / 10nM and without BHL, bacteria were inoculated at OD600nm=0.1.Then, fluorescence measurements of eGFP were performed for 15H (hourly in triplicata until 7H) in opaque 96-wells (FluoStars). For each curve, the first measurement point (T0) was set at 0 and subtracted to the following ones. In addition, a negative control E.coli Nissle wild type was also used (color)

Figure 1 eGFP expression according to the BHL concentration and over time. Different concentrations of BHL were performed to evaluate the effect of BHL in E. coli Nissle. Thanks to the BHL, the engineered E. coli should be able to express eGFP. Without BHL, no eGFP expression should be detected.

The Figure 1 shows the fluorescence intensity of each bacterial culture according to the time. While the wild type E.coli Nissle auto-fluorescence is used as blank, all the other transformed cultures show significant eGFP expression.

First, we can see that transformed E.coli Nissle produce low quantities of eGFP even without BHL. However, we can also observe that the more BHL, the more eGFP expression. Indeed, with 100µM and 10µM of BHL, after 3h of culture, the fluorescent intensity increases sharply up to 600 or more. Nevertheless, even if curves reach 400 and 100 with 1µM and 100nM respectively, weaker concentrations of BHL (10nM) don't allow transformed E.coli Nissle to significantly express eGFP.

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