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Team:Colombia/Project/Experiments/Ralstonia
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=Ralstonia Experiments= | =Ralstonia Experiments= | ||
| + | ==Ralstonia BioBricks== | ||
| + | We cloned and generate biobricks for the three genes (phcA, phcS and phcR) involved in the sensing of the Quorum Sensing signal 3 OH-PAME especific of ''Ralstonia solanacearum'', in addition, we cloned the promoter of xpsR (PxpsR) wich is responsive to the presence of 3 OH-PAME. Figure 1 shows enzymatic confirmation for all the four parts in the backbone pBS1C3. | ||
| + | |||
| + | ==xpsR Biosensor== | ||
| + | |||
==Response of ''PxpsR'' to synthetic 3-OH-PAME== | ==Response of ''PxpsR'' to synthetic 3-OH-PAME== | ||
'''Objective''' | '''Objective''' | ||
| - | To determine the response of PxpsR in ''Ralstonia Solanacearum'' | + | To determine the response of PxpsR in a mute ''Ralstonia Solanacearum'' to the presence of 3-0H-PAME at different concentrations, using YFP as a fluorescent reporter. |
'''Description''' | '''Description''' | ||
| - | The | + | The PxpsR-eYFP 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'' | + | The mute mutant of ''Ralstonia Solanacearum'', which has a mutation in the gene phcB (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. |
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Contents |
Ralstonia Experiments
Ralstonia BioBricks
We cloned and generate biobricks for the three genes (phcA, phcS and phcR) involved in the sensing of the Quorum Sensing signal 3 OH-PAME especific of Ralstonia solanacearum, in addition, we cloned the promoter of xpsR (PxpsR) wich is responsive to the presence of 3 OH-PAME. Figure 1 shows enzymatic confirmation for all the four parts in the backbone pBS1C3.
xpsR Biosensor
Response of PxpsR to synthetic 3-OH-PAME
Objective To determine the response of PxpsR in a mute Ralstonia Solanacearum to the presence of 3-0H-PAME at different concentrations, using YFP as a fluorescent reporter.
Description The PxpsR-eYFP 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, which has a mutation in the gene phcB (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.
