Difference between revisions of "Part:BBa K3771010"

Latest revision as of 03:56, 22 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

Fig. 1. Construction of OmpA/OprF chimeric protein

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, [3] 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.

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
2. Ortiz-Suarez Maite L, Samsudin F, Piggot Thomas J, Bond Peter J, Khalid S. Full-Length OmpA: Structure, Function, and Membrane Interactions Predicted by Molecular Dynamics Simulations. Biophysical Journal. 2016;111(8):1692-1702. doi:10.1016/j.bpj.2016.09.009
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 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]

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 <br><br><b style="font-size:1.3rem">Characterization
 </b>
-<br><br>The ompA/oprF sequence was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.
+<br><br>The <i>ompA/oprF</i> sequence was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.
 <html>
 <br>
 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 <img src="https://2021.igem.org/wiki/images/f/fd/T--NCKU_Tainan--pcr_ompA_oprF.gif.jpg" style="width:30%;">
-</div><p align="center">Fig. 2. Confirmation of our construction by PCR. M: Marker; Lane 1: ompA/oprF (1110 bp)</p>
+</div><p align="center">Fig. 2. Confirmation of our construction by PCR. M: Marker; Lane 1: <i>ompA/oprF</i> (1110 bp)</p>
 </html>
 <br><br>Expression of OmpA/OprF chimeric protein was confirmed by western blot using anti-OmpA antibody.