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Team:Colombia/Project/Experiments/Ralstonia
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'''Description''' | '''Description''' | ||
| - | The PxpsR+YFP biobrick was digested with XbaI and | + | The PxpsR+YFP biobrick was digested with XbaI and SpeI, and then was ligated to an 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, which 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, | + | 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, 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. |
Revision as of 00:52, 22 September 2012
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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. 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, which 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, 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.
