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Ralstonia Experiments

Response of PxpsR to synthetic 3-OH-PAME

Objective To determine the response of PxpsR in Ralstonia Solanacearum AW-1 to the presence of 3-0H-PAME at different concentrations, using YFP as a fluorescent reporter.

Description The PxpsR-yfp biobrick was digested with XbaI and SpeI, and then was ligated to an XbaI digested pML123 plasmid. Transformed colonies were confirmed via enzymatic digestions, to determine whether the biobrick had ligated in the opposite direction to the strong promoter npt (Figure 1). This way the biobrick would not be induced by the npt promoter present in the palsmid, and therefore, the fluorescent response due to the presence of 3-OH-PAME would depend solely on PxpsR. pML123 has a replication origin, called pVS1, which allows it to be replicated in Xanthomonas, Pseudomonas and Ralstonia. The mute mutant of Ralstonia Solanacearum AW-1, which has a mutation in 3-OH-PAME synthetase. Although it has the ability to sense 3-OH-PAME, Ralstonia's Quorum sensing signal, it cannot produce it, which allows to create an environment with a known concentration of 3-OH-PAME at all times, indepent of the number of bacterias present. This strain was transformed with the PxpsR-yfp::PML123 plasmid, and the fluorescent response was determined at different concentrations of synthetic 3-OH-PAME. Based on the fluorescent response, the inducibility of PxpsR was determined.