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.
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
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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