Other

Part:BBa_K4274005

Designed by: Jiawen Chen   Group: iGEM22_KEYSTONE   (2022-09-30)

PmanP

The manP promoter (PmanP) is situated in the upstream and same orientation of the mannose operon of Bacillus subtilis. It is reported that the presence of mannose could increase the beta-galactosidase activity, then result in a 4-to-7-fold increase in expression of lacZ (Sun et al., 2010). Besides, this promoter is not active in E. coli, but is activated by manR in Bacillus subtilis, which constitutes the mannose operon of B. subtilis. Based on it, the pJOE8999 plasmid system, which allows genome editing of B. subtilis, was designed to carry cas9 gene under the control of the B. subtilis mannose-inducible promoter PmanP (Altenbuchner et al., 2016). In this experiment, we are using a 0.2% concentration of mannose to induce cas9 expression of vector pJOE8999 to enable CRISPR-Cas9-based genome editing on B. subtilis strain 168.

Usage and Biology

The manP promoter (PmanP) is situated in the upstream and same orientation of the mannose operon of Bacillus subtilis. It is reported that the usage of this promoter will increase the expression of lacZ in B. subtilis for about 4 to 7 folds while using mannose as the carbon source. Therefore, this promoter was designed on pJOE8999 plasmid which is usually used for the gene-editing in B. subtilis genome. And with the help of pJOE8999, our team has succeeded in knocking-out/in genes after mannose-induction.

In the future, other iGEM teams can use this part regulate the heterologous expression of protein in B. subtilis and even the CRISPR-cas9 system for the gene-editing of B. subtilis.

Source

Bacillus subtilis

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]


References

[1] Sun T., Altenbuchner J. Characterization of a Mannose Utilization System in Bacillus subtilis. Journal of Bacteriology, 192(8), 2128-2139 (2010). https://doi.org/10.1128/JB.01673-09
[2] Altenbuchner J. Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system. Applied and Environmental Microbiology, 82(17), 5421-5427(2016). https://doi.org/10.1128/AEM.01453-16

[edit]
Categories
Parameters
None