Difference between revisions of "Part:BBa K3771009"
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<br>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 [1,10]. 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].<br>
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<br>The OmpA/OprF chimeric protein consists of amino acid sequences from both OmpA transmembrane protein from <i>E. coli</i> and OprF porin protein from <i>P. aeruginosa</i> [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].<br>
  
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to be edited
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<br><b style="font-size:1.3rem">Biology</b>
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://2021.igem.org/File:T--NCKU_Tainan--ompA_oprF.gif" style="width:35%;">
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</div>
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<p align="center">Figure 1: Construction of OmpA/OprF chimeric protein</p>
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<br>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 <i>E. coli</i> outer membrane, initiating the phage shock protein (Psp) system and its signal transduction to the inner membrane [3].<br>
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<br><b style="font-size:1.3rem">Usage</b>
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<br>
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<br>In the IFN-γ sensing system, OmpA/OprF chimeric protein expression is regulated by the <i>ompA</i> promoter. Binding of IFN-γ to the OmpA/OprF chimeric protein activates the <i>pspA</i> promoter, producing the enzyme required for the synthesis of taurine. <br>
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<br><b style="font-size:1.3rem">Characterization</b>
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<br>The <i>ompA/oprF</i> sequence was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.<br>
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://2021.igem.org/wiki/images/f/fd/T--NCKU_Tainan--pcr_ompA_oprF.gif.jpg" style="width:35%;">
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</div>
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<p align="center">圖片描述</p>
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<br>Expression of OmpA/OprF chimeric protein was confirmed by western blot using anti-OmpA antibody.<br>
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--results_ompA_wb.jpg" style="width:35%;">
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</div>
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<p align="center">圖片描述</p>
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<br><b style="font-size:1.3rem">References</b>
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<br>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
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https://pubmed.ncbi.nlm.nih.gov/11906175/
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<br>2. Wu L. Recognition of Host Immune Activation by Pseudomonas aeruginosa. Science. 2005;309(5735):774-777. doi:10.1126/science.1112422
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https://pubmed.ncbi.nlm.nih.gov/16051797/
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<br>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
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 12:15, 18 October 2021


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.

<img src="T--NCKU_Tainan--pcr_ompA_oprF.gif.jpg" style="width:35%;">

圖片描述


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

<img src="T--NCKU_Tainan--results_ompA_wb.jpg" style="width:35%;">

圖片描述



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]