Difference between revisions of "Part:BBa K4119078"
 
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     <p style="font-size: 180%; font-weight: bold;">Results:</p>
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     <p style="font-size: 160%; font-weight: bold;">Result:
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     <p>The expression level of Bs2 in the control of PvgbF3 under aerobic conditions was 1.8 higher than PvgbF3 under micro oxygen conditions</p>
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    </p>
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     <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/h2/img15.png" width="80%" height="80%"></p>
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     <p style="font-size: 160%; font-weight: bold;">Adjusting the distance between FNR binding site and the -35 region of
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        promoter vgb fine tunes the inhibitory effect of oxygen on the promoter.
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    </p>
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     <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/p5.png" width="80%" height="80%"></p>
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    <div align="center">
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        <strong>Fig.9 Construction of the recombinant plasmid for pMTL-PvgbF7-bs2</strong>
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    </div>
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    <p>By consulting the literature and consulting the authors [2], we learned that the distance between the FNR binding
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        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
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        reducing the interval speacer between-35 and-10 region to 17bp.
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        The plasmid pMTL-Pvgb-bs2 was extracted from recombined E. coli CA434 pMTL-Pvgb-bs2 constructed previously.
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        In this experiment, the megaprimers were obtained by PCR technique using the plasmid pMTL-Pvgb-bs2 as the
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        template, and then the mutant plasmid was obtained by PCR using those megaprimers to amplify the plasmid.
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    </p>
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    <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/p6.png" width="80%" height="80%"></p>
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    <div align="center">
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        <strong>Figure 10. Expression effect of pMTL-Pvgb-F7-bs2 at different oxygen concentratio</strong>
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    </div>
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    <p>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.
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        Under aerobic and microaerobic conditions, the modified promoter (Pvgb-F7) was separately 0.267 and 0.422 folds
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        of the controlled group (Pvgb). The modified promoter still has the regulatory effect brought by FNR and its
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        based oxygen-related biosensor system, which induction ratio increased to 6.28, compared with 3.97 of Pvgb as
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        control.
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    </p>
 
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Latest revision as of 15:14, 12 October 2022


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.


Document

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
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]