Part:BBa_K3771044

PpspA-CS-6xHis-PompA-OmpA/OprF

Description

This composite part is a component of the IFN-γ sensing system and was used to express the taurine production enzyme, L-cysteine sulfonic acid synthase (CS).

Biology

The ompA 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 CS-his-tag. CS converts o-phospho-l-serine into L-cysteine sulfonic acid in the taurine synthesis L-cysteine sulfinic acid pathway[2].

Fig.1. Taurine pathways in E. coli

Usage

We ligased the P_ompA-ompA/oprF fragment and P_pspA-cs on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.

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/

2. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093 https://pubmed.ncbi.nlm.nih.gov/30516051/

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 1915
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 591
  
• 1000
  COMPATIBLE WITH RFC[1000]