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<br><br>In our project, the sequence of OmpA protein was used to construct the OmpA/OprF chimeric protein.
 
<br><br>In our project, the sequence of OmpA protein was used to construct the OmpA/OprF chimeric protein.
  
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<br>To construct our OmpA/OprF chimeric protein, we replaced Loop 1 (AA 38-54) of OmpA from E. coli with Loop 5 (AA 198-237) of OprF from P. aeruginosa, [4] which resulted in the final OmpA/OprF chimeric protein. The new extracellular peptides of the OmpA/OprF chimeric protein allow for binding of IFN-γ to the E. coli outer membrane.
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<br><br>In the IFN-γ sensing system, OmpA/OprF chimeric protein expression is driven 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.
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<br><br><b style="font-size:1.3rem">Characterization
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                <a href="https://2021.igem.org/File:T--NCKU_Tainan--ompA_oprF.gif" target="_blank" style="width:60%"><img src="https://2021.igem.org/File:T--NCKU_Tainan--ompA_oprF.gif" alt="" title="" style="width:60%"></a>
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<br><br>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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<br><br>Expression of OmpA/OprF chimeric protein was confirmed by western blot using anti-OmpA antibody.
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Revision as of 07:53, 17 October 2021


Outer Membrane Protein A (OmpA)



Description

Outer membrane protein A (OmpA) is a 35 kDa transmembrane protein found in many enterobacteria. It is responsible for maintaining the stability of the bacterial membrane and cell signaling. [1]

Biology

In E. coli, the β-barrel conformation of OmpA is composed of extracellular loops that help play a role in the detection of outer membrane stress and binding of extracellular molecules. OmpA also serves as a porin channel that regulates water transport within the bacterial cell. [2]

Usage

In our project, the sequence of OmpA protein was used to construct the OmpA/OprF chimeric protein.


To construct our OmpA/OprF chimeric protein, we replaced Loop 1 (AA 38-54) of OmpA from E. coli with Loop 5 (AA 198-237) of OprF from P. aeruginosa, [4] which resulted in the final OmpA/OprF chimeric protein. The new extracellular peptides of the OmpA/OprF chimeric protein allow for binding of IFN-γ to the E. coli outer membrane.

In the IFN-γ sensing system, OmpA/OprF chimeric protein expression is driven 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.



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





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 881
    Illegal BamHI site found at 744
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]