Difference between revisions of "Part:BBa K3771009"
 
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<partinfo>BBa_K3771009 short</partinfo>
 
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<br><b style="font-size:1.3rem">Description</b>
 
<br><b style="font-size:1.3rem">Description</b>
 
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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 [3].<br>
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<br><b style="font-size:1.3rem">Biology</b>
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<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/a/af/T--NCKU_Tainan--ompA_oprF.gif" style="width:60%;">
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<p align="center">Fig. 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>
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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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<p align="center">Fig. 2. Confirmation of our construction by PCR. M: Marker; Lane 1: <i>ompA/oprF</i> (1110 bp)</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:50%;">
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<p align="center">Fig. 3. Confirmation of protein expression by western blot. Lane 1: OmpA positive control (~35 kDa); Lane 2: negative control; Lane 3, 4: OmpA/OprF chimeric protein (~39kDa)</p>
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<br><b style="font-size:1.3rem">References</b>
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<br>
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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
  
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===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 02:51, 20 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 [3].

Biology

Fig. 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.

Fig. 2. Confirmation of our construction by PCR. M: Marker; Lane 1: ompA/oprF (1110 bp)


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

Fig. 3. Confirmation of protein expression by western blot. Lane 1: OmpA positive control (~35 kDa); Lane 2: negative control; Lane 3, 4: OmpA/OprF chimeric protein (~39kDa)


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]