Part:BBa_K4119078
PvgbF3
PvgbF3 is an oxygen inducible promoter.PvgbF3 was modified based on the Pvgb constitutive promoter from Vitreoscilla.PvgbF3 were applied in E.coli successfully.
Result:
Adjusting the distance between FNR binding site and the -35 region of promoter vgb fine tunes the inhibitory effect of oxygen on the promoter.
By consulting the literature and consulting the authors [2], we learned that the distance between the FNR binding site and the -35 region of the promoter has a large impact on promoter transcriptional regulation. To adjust the distance of FNR binding site from the -35 region, we used site-directed mutagenesis. Megaprimer mutation technique was used to separate the FNR binding site from the -35 region by, changing the distance between the FNR binding site to 3bp and 7bp from -35 region, while changing the sequence from the second half of the FNR binding site and the -35 region to more conservative sequences [2,] with higher expressive effects, and reducing the interval speacer between-35 and-10 region to 17bp. The plasmid pMTL-Pvgb-bs2 was extracted from recombined E. coli CA434 pMTL-Pvgb-bs2 constructed previously. In this experiment, the megaprimers were obtained by PCR technique using the plasmid pMTL-Pvgb-bs2 as the template, and then the mutant plasmid was obtained by PCR using those megaprimers to amplify the plasmid.
By analyzing the fluorescence intensity data, it can be found that the increase in the distance between the FNR binding site and the -35 region of the promoter could result in a certain decrease in its expression effect. Under aerobic and microaerobic conditions, the modified promoter (Pvgb-F7) was separately 0.267 and 0.422 folds of the controlled group (Pvgb). The modified promoter still has the regulatory effect brought by FNR and its based oxygen-related biosensor system, which induction ratio increased to 6.28, compared with 3.97 of Pvgb as control.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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