Regulatory

Part:BBa_K4998028

Designed by: Rafail Andreou   Group: iGEM23_thessaloniki   (2023-10-09)

PtrC-2-derived promoter

Usage and Biology

This part contains the sequence of a strong, IPTG-inducible promoter that is suitable for gene expression in E. coli strains. The promoter sequence contains three operator sites that cause lactose molecules to bind tightly to them. The operator sites are 20-base long, inversed repeat sequences of the natural lactose operator’s first half. [1, 2]

IPTG induction

IPTG, or Isopropyl β-D-1-thiogalactopyranoside is a widely used molecular biology reagent. It is an analog of allolactose, the natural inducer of the lac operon. In E. coli, the lac operon consists of three genes, regulated by an operator region which, in the absence of lactose or the presence of glucose in the medium, is occupied by a lac repressor molecule, LacI, that prohibits the expression of the three genes. When present in the medium, IPTG binds to the repressor, in the same way as allolactose, and releases it from the operator region, enabling the three genes’ transcription. [3] This part is used in the iGEM Thessaloniki’s 2023 project, Euphoresis, as the promoter region of the TetR gene (BBa….). The TetR is part of the kill-switch mechanism developed for the Bacillus subtilis WB800N strain.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 37
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

References

[1] Sadler, J. R., Sasmor, H., & Betz, J. L. (1983). A perfectly symmetric lac operator binds the lac repressor very tightly. Proceedings of the National Academy of Sciences, 80(22), 6785–6789. https://doi.org/10.1073/pnas.80.22.6785

[2] Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2015). “Deadman” and “passcode” microbial kill switches for bacterial containment. Nature Chemical Biology, 12(2), 82–86. https://doi.org/10.1038/nchembio.1979

[3] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The impact of IPTG induction on plasmid stability and heterologous protein expression by escherichia coli biofilms. International Journal of Molecular Sciences, 21(2), 576. https://doi.org/10.3390/ijms21020576

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