Difference between revisions of "Part:BBa K3771009"

Revision as of 17:38, 19 October 2021 (view source) Evelynnn (Talk | contribs) ← Older edit		Revision as of 17:43, 19 October 2021 (view source) Evelynnn (Talk | contribs) Newer edit →
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	<img src="https://2021.igem.org/wiki/images/a/af/T--NCKU_Tainan--ompA_oprF.gif" style="width:60%;">		<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>	+	<p align="center">Fig. 1: Construction of OmpA/OprF chimeric protein</p>
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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%;">		<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>	+	<p align="center">Fig. 2. Confirmation of our construction by PCR. M: Marker; Lane 1: <i>ompA/oprF</i> (1110 bp)</p>
	<br>Expression of OmpA/OprF chimeric protein was confirmed by western blot using anti-OmpA antibody.<br>		<br>Expression of OmpA/OprF chimeric protein was confirmed by western blot using anti-OmpA antibody.<br>
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	<img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--results_ompA_wb.jpg" style="width:50%;">		<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>	+	<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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Revision as of 17:43, 19 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