Difference between revisions of "Part:BBa K4800001"
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         promoter vgb fine tunes the inhibitory effect of oxygen on the promoter.
 
         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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     <p><img src="https://static.igem.wiki/teams/4800/wiki/parts/mmcar.png" width="80%" height="80%"></p>
 
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         <strong>Fig.9 Construction of the recombinant plasmid for pMTL-PvgbF7-bs2</strong>
 
         <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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     <p>MmCAR is a carboxylic acid reductase from marine bacteria. During the experiment, we mutated the original 302nd amino
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        site and the -35 region of the promoter has a large impact on promoter transcriptional regulation.
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      acid glutamine to glutamic acid through acid-base mutation. The mutated Q302E has a significant improvement compared to  
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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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      the original one
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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><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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    <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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Revision as of 11:38, 4 October 2023


rrnB T1 terminator

Carboxylate reductase from Mycobacterium marinum(Mutation of glutamine at position 302 to glutamate)



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

MmCAR is a carboxylic acid reductase from marine bacteria. During the experiment, we mutated the original 302nd amino acid glutamine to glutamic acid through acid-base mutation. The mutated Q302E has a significant improvement compared to the original one

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 357
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 357
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 357
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 357
  • 1000
    COMPATIBLE WITH RFC[1000]