Part:BBa_K4119079

Pvgb-F7-bs2

We use fluorescent protein Bs2 to evaluate the promoter strength of Pvgb-F7.

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.

Fig.9 Construction of the recombinant plasmid for pMTL-PvgbF7-bs2

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.

Figure 10. Expression effect of pMTL-Pvgb-F7-bs2 at different oxygen concentratio

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 361
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 361
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 361
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 361
  
• 1000
  COMPATIBLE WITH RFC[1000]