Difference between revisions of "Part:BBa K2771030:Experience"
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<h2>USP-Brazil Characterization</h2> | <h2>USP-Brazil Characterization</h2> | ||
| − | This part has been tested alongside with six quorum sensing promoters, pLux ([https://parts.igem.org/Part:BBa_K2771021 BBa_K2771021]), pLas ([https://parts.igem.org/Part:BBa_K2771022 BBa_K2771022]), pRhl([https://parts.igem.org/Part:BBa_K2771023 BBa_K2771023]), pCin([https://parts.igem.org/Part:BBa_K2771024 BBa_K2771024]), pTra ([https://parts.igem.org/Part:BBa_K2771025 BBa_K2771025]) and pRpa [https://parts.igem.org/Part:BBa_K2771026 BBa_K2771026]. Assay in 2mL LB in 24-well plate, shaking at 180rpm and 37ºC, 2.5 mM arabinose, taking samples every 3hrs: | + | This part has been tested alongside with six quorum sensing promoters, pLux ([https://parts.igem.org/Part:BBa_K2771021 BBa_K2771021]), pLas ([https://parts.igem.org/Part:BBa_K2771022 BBa_K2771022]), pRhl([https://parts.igem.org/Part:BBa_K2771023 BBa_K2771023]), pCin([https://parts.igem.org/Part:BBa_K2771024 BBa_K2771024]), pTra ([https://parts.igem.org/Part:BBa_K2771025 BBa_K2771025]) and pRpa ([https://parts.igem.org/Part:BBa_K2771026 BBa_K2771026]). Assay in 2mL LB in 24-well plate, shaking at 180rpm and 37ºC, 2.5 mM arabinose, taking samples every 3hrs. Before measuring each sample, we centrifuged the cells and resuspended the sample in H2O, to eliminate LB autofluorescence. Note that Y axis is in ratiometric activity, because reporter is measured as a ratio of YFP/CFP fluorescence. For excitation-emission wavelenghts, we used 430-480 for CFP and 500-530 for YFP: |
| + | [[File:T--USP-Brazil--Lux_Las_assay.png|500px|thumb|none|alt=experiment 1.]] | ||
| + | [[File:T--USP-Brazil--Others assay.png|500px|thumb|none|alt=experiment 2.|Figures 1 and 2: Labels refer as Sender plasmid:Receiver plasmid, combining BBa_K2771030 with different quorum sensing promoters]] | ||
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| + | We also did an assay comparing activity when induced by arabinose and when induced directly by 10<sup>-4</sup>mM HSL. Auto-inducing showed slower response, but much higher threshold (levels and speeds slower than in the last graphs due to growing in M9 media instead of LB: | ||
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| + | [[File:T--USP-Brazil--ara HSL assay.png|500px|thumb|none|alt=experiment 3.]] | ||
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| + | When testing the promoter of tha Las system, however, it only showed activity similar to what's seen with LB with the synthetic HSL induction, what leads us to believe that the culture couldn't reach a concentration of 10<sup>-4</sup>mM HSL with the synthase in M9 medium: | ||
| + | [[File:T--USP-Brazil--Lux Las 1 17 10.png|400px|thumb|none|alt=experiment 3.]] | ||
| + | [[File:T--USP-Brazil--Lux Las 2 17 10.png|400px|thumb|none|alt=experiment 3.]] | ||
===Applications of BBa_K2771030=== | ===Applications of BBa_K2771030=== | ||
Latest revision as of 23:16, 17 October 2018
USP-Brazil Characterization
This part has been tested alongside with six quorum sensing promoters, pLux (BBa_K2771021), pLas (BBa_K2771022), pRhl(BBa_K2771023), pCin(BBa_K2771024), pTra (BBa_K2771025) and pRpa (BBa_K2771026). Assay in 2mL LB in 24-well plate, shaking at 180rpm and 37ºC, 2.5 mM arabinose, taking samples every 3hrs. Before measuring each sample, we centrifuged the cells and resuspended the sample in H2O, to eliminate LB autofluorescence. Note that Y axis is in ratiometric activity, because reporter is measured as a ratio of YFP/CFP fluorescence. For excitation-emission wavelenghts, we used 430-480 for CFP and 500-530 for YFP:
We also did an assay comparing activity when induced by arabinose and when induced directly by 10-4mM HSL. Auto-inducing showed slower response, but much higher threshold (levels and speeds slower than in the last graphs due to growing in M9 media instead of LB:
When testing the promoter of tha Las system, however, it only showed activity similar to what's seen with LB with the synthetic HSL induction, what leads us to believe that the culture couldn't reach a concentration of 10-4mM HSL with the synthase in M9 medium:
Applications of BBa_K2771030
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