Difference between revisions of "Part:BBa K3771010"
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<br><br><b style="font-size:1.3rem">Biology | <br><br><b style="font-size:1.3rem">Biology | ||
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− | <br><br>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] | + | <br><br>In <i>E. coli</i>, 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] |
<br><br><b style="font-size:1.3rem">Usage | <br><br><b style="font-size:1.3rem">Usage | ||
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− | <div | + | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> |
− | + | <img src="https://2021.igem.org/wiki/images/a/af/T--NCKU_Tainan--ompA_oprF.gif" style="width:50%;"> | |
− | + | </div> | |
− | + | <p align="center">Fig. 1. Construction of OmpA/OprF chimeric protein | |
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+ | </p> | ||
+ | </html> | ||
+ | <br>In our project, the sequence of OmpA protein was used to construct the OmpA/OprF chimeric protein. | ||
+ | <br>To construct our OmpA/OprF chimeric protein, we replaced Loop 1 (AA 38-54) of OmpA from <i>E. coli</i> with Loop 5 (AA 198-237) of OprF from <i>P. aeruginosa</i>, [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 <i>E. coli</i> outer membrane. | ||
+ | <br>In the IFN-γ sensing system, OmpA/OprF chimeric protein expression is driven 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><br><b style="font-size:1.3rem">Characterization | ||
+ | </b> | ||
+ | <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: <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. | ||
+ | <html> | ||
+ | <div style="width=100%; display:flex; align-items: center; justify-content: center;"> | ||
+ | <img src="https://2021.igem.org/wiki/images/9/9b/T--NCKU_Tainan--results_ompA_wb.jpg" style="width:50%;"> | ||
+ | </div> | ||
+ | <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> | ||
+ | </html> | ||
+ | <br><br><b style="font-size:1.3rem">References | ||
+ | </b> | ||
+ | <br> | ||
+ | <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 | ||
+ | <br>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 | ||
+ | <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 | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 881
Illegal BamHI site found at 744 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]