Difference between revisions of "Part:BBa K4800001"
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| + | <title>Document</title> | ||
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| − | < | + | <p style="font-size: 160%; font-weight: bold;">Result: |
| − | + | </p> | |
| − | < | + | <p style="font-size: 160%; font-weight: bold;">Adjusting the distance between FNR binding site and the -35 region of |
| − | 2- | + | promoter vgb fine tunes the inhibitory effect of oxygen on the promoter. |
| − | < | + | </p> |
| − | + | <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/p5.png" width="80%" height="80%"></p> | |
| − | < | + | <div align="center"> |
| − | + | <strong>Fig.9 Construction of the recombinant plasmid for pMTL-PvgbF7-bs2</strong> | |
| + | </div> | ||
| + | <p>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. | ||
| + | </p> | ||
| + | <p><img src="https://static.igem.wiki/teams/4119/wiki/jt-files/p6.png" width="80%" height="80%"></p> | ||
| + | <div align="center"> | ||
| + | <strong>Figure 10. Expression effect of pMTL-Pvgb-F7-bs2 at different oxygen concentratio</strong> | ||
| + | </div> | ||
| + | <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. | ||
| + | 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. | ||
| + | </p> | ||
| + | </body> | ||
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| − | + | <span class='h3bb'>Sequence and Features</span> | |
| − | + | <partinfo>BBa_K4119080 SequenceAndFeatures</partinfo> | |
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Revision as of 11:18, 4 October 2023
rrnB T1 terminator
Carboxylate reductase from Mycobacterium marinum(Mutation of glutamine at position 302 to glutamate)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
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.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 357
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 357
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 357
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 357
- 1000COMPATIBLE WITH RFC[1000]