Difference between revisions of "Part:BBa K4119024"
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     <p style="font-size: 180%; font-weight: bold;">Results:</p>
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     <p style="font-size: 180%; font-weight: bold;">Result:
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     <p>The inhibition effect of the improved promoter(P2vgb) was stronger than that of the control group(Pvgb) under aerobic conditions. Under the induction of microaerobic conditions, the improved promoter(P2vgb) behaves similarly to the control group(Pvgb) under aerobic conditions. This suggests that tandem promoters improve protein expression.</p>
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     <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/h2/img10.png" width="80%" height="80%"></p>
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     <p style="font-size: 160%; font-weight: bold;">Doubling promoter vgb enhances the expression level of the promoter.
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    </p>
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    <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/p1.png" width="80%" height="80%"></p>
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    <div align="center">
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        <strong>Fig.5 Construction of the recombinant plasmid for pMTL- P2vgb-bs2</strong>
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    </div>
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    <p>Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not
 +
        ideal, so we tried to enhance its ability to express proteins by doubling promoter vgb.
 +
        We decided to insert a repeated Pvgb fragment into the upstream of Pvgb , hoping that the expression of Bs2
 +
        fluorescent protein could be improved.
 +
        We have converted the construction and sequence analysis towards the recombinant plasmid into E. coli CA434, but
 +
        time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C.
 +
        tyrobutyricum by conjugation, so the detection of fluorescence intensity data were carried out in E. coli to
 +
        verify the effectiveness of the improvements for the vgb promoter.
 +
        By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid
 +
        pMTL- P2vgb-bs2 together with Pvgb-bs2 as control, under those two conditions respectively, and sampled the
 +
        culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment.
 +
    </p>
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     <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/p2.png" width="80%" height="80%"></p>
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    <div align="center">
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        <strong>Figure 6. Expression effect of pMTL-P2vgb-bs2 at different oxygen concentrations</strong>
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    </div>
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    <p>After collating and analyzing the fluorescence intensity data (Figure 6), it can be seen obviously that the
 +
        inhibition effect of the improved promoter(P2vgb) was stronger than that of the control group(Pvgb) under
 +
        aerobic conditions. Under the induction of microaerobic conditions, the improved promoter(P2vgb) behaves
 +
        similarly to the control group(Pvgb) under aerobic conditions. This suggests that tandem promoters improve
 +
        protein expression.</p>
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Revision as of 15:06, 12 October 2022


Pvgb-double promoter->Bs2->Cpa fdx terminator

it's Pvgb-double promoter->Bs2->Cpa fdx terminator


Document

Result:

Doubling promoter vgb enhances the expression level of the promoter.

Fig.5 Construction of the recombinant plasmid for pMTL- P2vgb-bs2

Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not ideal, so we tried to enhance its ability to express proteins by doubling promoter vgb. We decided to insert a repeated Pvgb fragment into the upstream of Pvgb , hoping that the expression of Bs2 fluorescent protein could be improved. We have converted the construction and sequence analysis towards the recombinant plasmid into E. coli CA434, but time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C. tyrobutyricum by conjugation, so the detection of fluorescence intensity data were carried out in E. coli to verify the effectiveness of the improvements for the vgb promoter. By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid pMTL- P2vgb-bs2 together with Pvgb-bs2 as control, under those two conditions respectively, and sampled the culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment.

Figure 6. Expression effect of pMTL-P2vgb-bs2 at different oxygen concentrations

After collating and analyzing the fluorescence intensity data (Figure 6), it can be seen obviously that the inhibition effect of the improved promoter(P2vgb) was stronger than that of the control group(Pvgb) under aerobic conditions. Under the induction of microaerobic conditions, the improved promoter(P2vgb) behaves similarly to the control group(Pvgb) under aerobic conditions. This suggests that tandem promoters improve protein expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 485
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 485
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 485
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 485
  • 1000
    COMPATIBLE WITH RFC[1000]