Team:Grenoble-Alpes/Parts

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PARTS

This year we created three different RFC10 compatible biobricks : two basics and one composite. These biobricks were made to be used as detectors in our system when inserted in the pSB1C3 backbone:

The first biobrick, BBa_K2629000, is a fragment of ProC gene from Pseudomonas Aeruginosa, which represents the probe for the bacterium’s detection. Introduced in a pSB1C3 backbone carrying BBa_J04450, it allows us to detect the presence of the bacterium, with a transformation in TOP10 E. Coli to express the reporter gene and observe its red fluorescence.
Sadly, we didn’t manage to insert this biobrick in the plasmid backbone, as our sequencing results were not showing any insertion at all (link to biobrick page on registry).
The second biobrick, BBa_K2629001, is a fragment of GyrA gene from Pseudomonas Aeruginosa carrying two mutations which are known to generate resistance to fluoroquinolones, which represents the probe for the resistance to this antibiotic. Introduced in a pSB1C3 backbone carrying BBaa_J4450, it allows us to detect the resistance of Pseudomonas Aeruginosa to fluoroquinolones, by expressing a red fluorescence in the transformed bacteria.
Regretfully, we haven’t get nice sensitivity results and thus we were not able to proceed with specificity tests, because of a bad activation of the plasmid probe (digestions that probably didn’t work well).
The third biobrick, BBa_K2629003, is a composite part. Our goal was to insert the BBa_K2629001 biobrick probe with a reporter gene derived from BBa_J04450, but which would produce BFP instead of RFP. We thus tried to create a biobrick combining BBa_K2629001 and the promoter-RBS-terminator sequences from BBa_J04450 with the BFP gene coming from BBa_K292015. This new biobrick would allow us to detect in the same time the bacterium and its resistance, with two different fluorescent signals.
Unfortunately, sequencing results told us we managed to replace RFP gene by the BFP one, but not to insert the probe gene. Due to the lack of time to finish the project, we didn’t manage to characterize this part.

In the end, we could only present two biobrick : BBa_K2629001 the antibiotic resistance detection, and BBa_K2629001, only with the modified BBa_J04450 expressing RFP instead of BFP. Furthermore, we lacked time to completely characterize these biobricks.