Part:BBa_J36836
Outer membrane protein A, aa 46-66
This part codes for amino acids #46-66 of outer membrane protein A (OmpA). It corresponds to a single transmembrane domain of OmpA, crossing the outer membrane of E. coli.
This was fused with the lipoprotein signal peptide upstream and streptavidin downstream in the project to express streptavidin on the outer surface of E. coli.
Contribution
- Group: Nanjing-China
- Author: Peiqing Sun, Kuisong Song, Wenyin Su
- Summary: We cloned and characterized OmpA and fused it with a heavy metal binding protein called PbrR. We successfully extended the application of OmpA into the cell surface display system of E.coli.
- Group: Nanjing-China
- Author: Bradley, Avery
- Summary: We performed a literature review of OmpA.
Usage and Biology
As we all know, improving the function or characterization of previously existing parts in the part registry of iGEM is not only important for maintenance of the part registry, but essential for other teams to utilize the part properly as well. Part BBa_J36836 encodes outer membrane protein (OmpA) of E. coli, which corresponds to a single transmembrane domain of OmpA. As far as we are concerned, this protein can be applied to the cell surface display system, which is to fix other proteins onto the surface of E.coli. This year we extended the application of OmpA in the construction of our artificial PS system. In our project, a metal binding protein PbrR is fused with OmpA so as to induce precipitation of CdS nanoparticles on the surface of E.coli cells. Protein PbrR is a special metal protein found in Cupriavidus metallidurans that specifically binds to Pb2+ ions. In realistic research we further examined that this protein also bears a high affinity to Cd2+ ions. We demonstrated in our project that when we fused PbrR with OmpA, the binding of PbrR with Cd2+ is greatly enhanced. (Figure 1).
Characterisation of OmpA
Protein PbrR is a special metal protein found in Cupriavidus metallidurans that specifically binds to Pb2+ ions. In realistic research we further examined that this protein also bears a high affinity to Cd2+ ions(Figure 1). We demonstrated in our project that when we fused PbrR with OmpA, the binding of PbrR with Cd2+ is greatly enhanced.
For organism M.thermoacetica, this kind of bacteria can produce S2- ions from cysteine and forms a higher sulfur concentration around the cell which then induces the precipitation of CdS nanoparticles when Cd2+ ions are added into the media. We assume that if we form a same local high concentration of Cd2+ with fused protein OmpA-PbrR on the outer cell membrane, we can also achieve a similar precipitation of CdS nanoparticles on to the walls of E.coli, the well model bacteria. To confirm the capability of our CdS system based on OmpA-PbrR, we conducted the same photo-catalytic assay. Bacteria were divided into three groups. Bacteria were induced to express OmpA-PbrR protein and cultured with both Cd2+ and S2- in the experiment group. Groups that either lacked induced expression or necessary ions to build semiconductors were negative controls. We found that illumination resulted in a same increasing trend in experiment group (Figure 2). This confirmed the photo-catalytic capability of our PbrR-based precipitation of semiconductors.
We also did a TEM imaging of the CdS particles formed on bacteria surface and demonstrated that the CdS particles are nanoparticles. It indicated that OmpA works efficiently on the cell surface display system of E.coli. To conclude, we utilized the part BBa_J36836 and extended its application to the cell surface display system in E.coli. We successfully displayed a kind of metal binding protein PbrR to the surface of E.coli and identified enhanced function of PbrR after fusion with OmpA. To learn more details about our project design, please see the following project overview part.
Usage and Biology
Characterization of OmpA OmpA, an outer membrane protein, consists of 325 residues (1). This protein consists of a N-terminal region with an eight stranded beta-barrel that anchors four loops which extend into the extracellular area (2). A fifteen-nucleotide long region of this protein connects the N-terminal region to the C-terminal region, which is located in the periplasm (3). OmpA has been shown to form dimers and it is hypothesized to exist in a hybrid state between its monomer and dimer forms (3). While there is conclusive evidence of the existence of the OmpA dimer, the function of this dimer is unclear (3, 4). OmpA has been shown to be heat-modifiable and its molecular mass ranges from 28 kDa to 36 kDa based on the temperature of the protein before the weight has been calculated. There are about 100,000 copies of OmpA per cell (2,5). OmpA is most commonly located in gram-negative bacteria and is best characterized in E. coli (5). However, OmpA has homologs in many different species such as P. aeruginosa and C. trachomatis (5). OmpA transcription has been shown to be dependent on environmental conditions (2). Factors such as nitrogen availability, adhesion to surfaces, and growth rate all impact the transcription of OmpA (6,7,8). For example, when the cell is growing at a higher rate, it maintains a mRNA half-life of about 15 minutes; however, with a slower growth rate, the mRNA half-life decreases to about 4 minutes (9). OmpA has many functions within E. coli. OmpA assists the cell in holding together the outer membrane and the peptidoglycan layer (1). While there has been documented nucleotide variability within the loop portion of the protein, the beta-barrel region remains consistent across many different E. coli species, hinting at this region's role in structural support (1). Further, these beta-barrels also play an important role in regulating environmental stress. When mutant bacterial strains without OmpA were formed, they showed greater responses to environmental stress and a greater death rate when exposed to acidic environments or osmotic shock compared to bacterial strains containing OmpA (1). In addition to providing E. coli with structural support, OmpA acts as a non-specific porin with a pore size of roughly 1 nm (10). OmpA and the Immune System While OmpA has many functions in E. coli, it plays a large role in the pathogenesis of E. coli. OmpA is simultaneously a way by which pathogenic bacteria can avoid host immune system defenses and become a target of the immune system (2,5). OmpA provides bacterial cells with methods to avoid the immune system. By providing a binding site for protein C4, OmpA is thought to avoid antibody detection (5). Further, OmpA enables E. coli cells to enter macrophages, replicate, and eventually lyse the macrophages (11). While OmpA helps the cell to avoid immune system responses, it is also an identifier of infection and triggers an immune response. For example, the immune system uses an enzyme called neutrophil elastase to destroy pathogenic bacteria (2,5). Normally, neutrophil elastase is able to effectively kill E. coli. However, in mutant bacterial strains without OmpA, neutrophil elastase is no longer effective, suggesting that OmpA acts as a target of the immune system (12). Furthermore, OmpA is thought to bind to a receptor on macrophages, alerting the infected cell to a potential bacterial invasion (13). OmpA as a Potential Vaccine Because of its role in pathogenesis, many scientists have considered the use of OmpA in creating a protein-based vaccine (14,15,16). Gu et al. demonstrated that each loop of OmpA produces an immune response and that each loop has specific antibodies that target it (15). Due to the strong immune response against OmpA, vaccines have been developed using this protein (14,15,16). These vaccines have been shown to decrease bacterial load in the lungs and provide higher levels of antigen-specific antibodies, supporting further research into these vaccines (14,15). The OmpA protein is highly conserved across many different bacterial strains (2). However, there are 22 different known polymorphisms in OmpA sequences (17). Despite the existence of these polymorphisms, OmpA vaccines are able to provide protection against many different strains of bacteria (15). OmpA and Synthetic Biology Applications While the N-terminal region of OmpA is highly conserved, the four loops exhibit high variability (1). This variability allows all four of the loops of OmpA to be modified in order to express a different protein (18, 19, 20, 21). By replacing the loop regions of OmpA with other DNA sequences, scientists are able to display heterologous proteins (19). Scientists have used the Lpp-OmpA system to help display these proteins (23, 24). This system consists of a fusion between the Lpp protein, the OmpA protein, and an additional protein, which varies based on the end-goal of the system (24). For example, researchers Scott et. al fused the human O6-alkylguanine DNA alkyltransferase protein, also known as SNAP, into this system (25). The SNAP protein is able to respond to the presence of benzylguanine, allowing it to act as a sensor (25). When the Lpp-OmpA-SNAP sensor is exposed to benzylguanine, it causes the surface of the E. coli to glow (25). This chimeric protein has wide applications in synthetic biology as it can act as a reporter gene (25). Another version of the Lpp-OmpA system was developed by Fasehee et al. (23). This version fused LPQTG (a sortase cleaving sequence), metallothionein, and the chitin binding domain (ChBD) to the Lpp-OmpA system. This fusion allowed metallothionein and ChBD to be expressed on the cell surface of E. coli. When srtA was activated, it cleaved the fused protein at the LPQTG site, releasing metallothionein and ChBD. Fasehee et al. were able to find evidence for the cleaving of this enzyme through the use of gel electrophoresis by comparing the results before and after the addition of srtA. Using this method, scientists can easily produce and purify recombinant proteins (23). While the Lpp-OmpA system has a myriad of applications, OmpA can be used in conjunction with other proteins as well. Aurand and March created chimeric OmpA proteins that are able to sense the presence of inflammatory mediators interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) (26). This protein consists of OmpA, which is naturally found in E. coli, with some of its extracellular loops replaced by OprF, which is endogenous to P. aeruginosa. When creating their chimeric sensors, Aurand and March tested various combinations of OmpA and OprF loops. They found that the chimeric proteins with loop one of OmpA replaced by loop 5 of OprF was best able to detect the presence of IFN-gamma. When loops 1 and 2 were replaced with loops 5 and 6, respectively, the system was able to detect levels of TNF-a at 150 pM (26). 1. Wang Y. The Function of OmpA in Escherichia coli. Biochemical and Biophysical Research Communications. 2002; 292 (2):396-401. 2. Smith SG, Mahon V, Lambert MA, Fagan RP. A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol Lett. 2007;273(1):1–11. 3. Ortiz-Suarez, M., Samsudin, F., Piggot, T., Bond, P., and Khalid, S. Full-Length OmpA: Structure, Function, and Membrane Interactions Predicted by MolecularDynamics Simulations. Biophysical Journal. 2016; 111: 1692-1702. 4. Kaspersen, J., Jessen, C., Vad, B., Sorensen, E., Andersen, K., Glasius, M., Oliveira, C., Otzen, D., Pedersen, J. Low‐Resolution Structures of OmpA⋅DDM Protein–Detergent Complexes. ChemBioChem. 2014; 15(14). 5. Confer, A., Ayalew, S. The OmpA family of proteins: roles in bacterial pathogenesis and immunity. Vet Microbiol. 2013;163(3-4):207-222. 6. Baev MV, Baev D, Radek AJ & Campbell JW. Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays. Appl Microbiol Biotechnol. 2006; 71: 317–322. 7. Otto K, Norbeck J, Larsson T, Karlsson KA & Hermansson M. Adhesion of type 1-fimbriated Escherichia coli to abiotic surfaces leads to altered composition of outer membrane proteins. J Bacteriol. 2001; 183: 2445–2453. 8. Lugtenberg B, Peters R, Bernheimer H & Berendsen W. Influence of cultural conditions and mutations on the composition of the outer membrane proteins of Escherichia coli. Mol Gen Genet. 1976;147: 251–262. 9. Nilsson G, Belasco JG, Cohen SN & von Gabain A. Growth-rate dependent regulation of mRNA stability in Escherichia coli. Nature. 1984;312: 75–77. 10. Sugawara E & Nikaido H. Pore-forming activity of OmpA protein of Escherichia coli. J Biol Chem. 1992; 267: 2507–2511. 11. Sukumaran SK, Shimada H & Prasadarao NV. Entry and intracellular replication of Escherichia coli K1 in macrophages require expression of outer membrane protein A. Infect Immun. 2003; 71: 5951–5961. 12. Belaaouaj A, Kim KS & Shapiro SD. Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 2000; 289: 1185–1188. 13. Soulas et. al. Cutting Edge: Outer Membrane Protein A (OmpA) Binds to and Activates Human Macrophages. The Journal of Immunology 2000; 165: 2335-2340. (fix citation numbers after here) 14. Lei et. al. DNA vaccine encoding OmpA and Pal from Acinetobacter baumannii efficiently protects mice against pulmonary infection, Molecular Biology Reports. 2019; 46: 5397-5408. 15. Gu et. al. Rational Design and Evaluation of an Artificial Escherichia coli K1 Protein Vaccine Candidate Based on the Structure of OmpA. Frontiers in Cellular and Infection Microbiology. 2018; 8:172. 16. Lin L, Tan B, Pantapalangkoor P, Ho T, Hujer AM et al. Acinetobacter baumannii rOmpA vaccine dose alters immune polarization and immunodominant epitopes. Vaccine. 2019;31:313–318. 17. Nielson et. al. Outer membrane protein A (OmpA) of extraintestinal pathogenic Escherichia coli. BMC Research Notes. 2020; 13:51. 18. Freudl et. al. Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. Journal of Molecular Biology. 1986; 188(3):491-494. 19. Freudl, R., Insertion of peptides into cell-surface-exposed areas of the Escherichia coli OmpA protein does not interfere with export and membrane assembly. Gene. 1989; 82(2): 229-236. 20. Pistor, S., Hobom, G. Expression of viral hemagglutinin on the surface ofE. coli . Klin Wochenschr 1998; 66: 110–116. 21. Schor et. al. Surface expression of malarial antigens in Salmonella typhimurium: induction of serum antibody response upon oral vaccination of mice. Vaccine. 1991; 9(9):675-681 22. Samuelson et. al. Display of proteins on bacteria. Journal of Biotechnology. 2002; 96:129-154. 23. Fasehee et. al. Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins. AMB Express. 2018; 8:107 24. Georgiou et. al. Display of pMactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-pMactamase fusions. Protein Engineering. 1996; 9(2):239-247. 25. Scott, Felicia Y. (2015). Surface Displayed SNAP as a New Reporter in Synthetic Biology [Master of Science In Biological Systems Engineering, Virginia Polytechnic Institute]. 26. Aurand and March. Development of a Synthetic Receptor Protein for Sensing Inflammatory Mediators Interferon-g andTumor Necrosis Factor-a. Biotechnology and Bioengineering. 2015; 113(3): 492-500.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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