Part:BBa_K3771017

PpspA-OFP-PlacI-OmpA/OprF

Description

This composite part is a component of the IFN-γ sensing system and was used in the IFN-γ induction assay, testing the efficiency of the pspA promoter.

Biology

The lacI promoter facilitates the constitutive expression of OmpA/OprF. Binding of IFN-γ to the OmpA/OprF chimeric protein induces the response of the phage shock protein (Psp) system, a highly conserved stress response system in enterobacteria [1]. Signal transduction from the outer membrane to the inner membrane activates the pspA promoter, initiating expression of OFP.

Usage

We ligased the P_lacI-ompA/oprF fragment and P_pspA-ofp on the pUC expression vector and transformed it into DH5α to complete construction of the plasmid. Then, the plasmid was purified and transformed into E. coli ompA mutant strain JW0940.

Characterization

Double digestion results are shown in Fig. 1.

Fig. 1. Double digestion of P_pspA-ofp-P_lacI-ompA/oprF

To test the function of the pspA promoter, OFP expression regulated by the induction of the pspA promoter was observed and recorded using a microplate reader. ompA promoter and lacI promoter activity levels were also compared.

Fig. 2. OFP expression of P_pspA-ofp-P_lacI-ompA/oprF

Fig. 3. OFP expression of P_pspA-ofp-P_ompA-ompA/oprF

References

1. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.x https://pubmed.ncbi.nlm.nih.gov/16045608/
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 12
  Illegal BamHI site found at 2055
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]