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| − | Carboxylate reductase from Mycobacterium marinum(Mutation of glutamine at position 302 to glutamate)
| + | In the pDONR vector series it protects the cloned gene from expression by vector-encoded promoters, thereby reducing possible toxicity. |
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| − | <p style="font-size: 160%; font-weight: bold;">Result:
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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><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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| − | <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.
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| − | To adjust the distance of FNR binding site from the -35 region, we used site-directed mutagenesis. Megaprimer
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| − | mutation technique was used to separate the FNR binding site from the -35 region by, changing the distance
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| − | between the FNR binding site to 3bp and 7bp from -35 region, while changing the sequence from the second half of
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| − | 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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| − | <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
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| − | 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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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
| − | <partinfo>BBa_K4119080 SequenceAndFeatures</partinfo> | + | <partinfo>BBa_K4800001 SequenceAndFeatures</partinfo> |
In the pDONR vector series it protects the cloned gene from expression by vector-encoded promoters, thereby reducing possible toxicity.
