From 2012.igem.org

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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 Spe I, and then was ligated to a XbaI digested PML123 plasmid. This way the biobrick was not 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, whch allows it to be replicated in Xanthomonas, Pseudomonas and Ralstonia. Ralstonia Solanacearum AW-1 is a strain of Ralstonia which has a mutation in 3-OH-PAME synthetase, making it a mute cell. Although it has the ability to sense 3-OH-PAME, Ralstonias Quorum sensing signal, it cannot produce it. This strain was transformed with the PxpsR+YFP+PML123 plasmid, and the fluorescent response was determined at different concentrations of synthetic 3-OH-PAME.