Coding

Part:BBa_K3771009

Designed by: KAO, YI-CHING   Group: iGEM21_NCKU_Tainan   (2021-10-01)
Revision as of 12:17, 18 October 2021 by Evelynnn (Talk | contribs)


OmpA/OprF chimeric protein

Description

The OmpA/OprF chimeric protein consists of amino acid sequences from both OmpA transmembrane protein from E. coli and OprF porin protein from P. aeruginosa [1,2]. The newly added loop from OprF allows OmpA/OprF chimeric protein to bind to IFN-γ and initiate a signal transduction to the inner membrane [4].

Biology

Figure 1: Construction of OmpA/OprF chimeric protein


One of the innate functions of OprF is the capability to bind and respond to IFN-γ. Of the four β-barrel loops in OmpA, the first loop is removed and replaced by a loop from OprF, resulting in an OmpA protein with a section of OprF protein. The new extracellular peptides of the OmpA/OprF chimeric protein allows for binding of IFN-γ to the E. coli outer membrane, initiating the phage shock protein (Psp) system and its signal transduction to the inner membrane [3].

Usage

In the IFN-γ sensing system, OmpA/OprF chimeric protein expression is regulated by the ompA promoter. Binding of IFN-γ to the OmpA/OprF chimeric protein activates the pspA promoter, producing the enzyme required for the synthesis of taurine.

Characterization

The ompA/oprF sequence was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.

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Expression of OmpA/OprF chimeric protein was confirmed by western blot using anti-OmpA antibody.

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References
1. Wang Y. The Function of OmpA in Escherichia coli. Biochemical and Biophysical Research Communications. 2002;292(2):396-401. doi:10.1006/bbrc.2002.6657 https://pubmed.ncbi.nlm.nih.gov/11906175/
2. Wu L. Recognition of Host Immune Activation by Pseudomonas aeruginosa. Science. 2005;309(5735):774-777. doi:10.1126/science.1112422 https://pubmed.ncbi.nlm.nih.gov/16051797/
3. Aurand TC, March JC. Development of a synthetic receptor protein for sensing inflammatory mediators interferon‐γ and tumor necrosis factor‐α. Biotechnology and Bioengineering. 2016;113(3):492-500. doi:10.1002/bit.25832 Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 813
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None