Part:BBa_K3739021
OmpA-GFP
This is an anchored proteins onto membranes through OmpA and use GFP to comformation. We use BBa_K3739049 and GFP to verify OmpA' s function which can anchor proteins onto membranes.
Biology
OmpA is an anchor protein from E. coli, which can anchor its passenger protein to the cell membrane. It has been widely used in cell-surface display. GFP is green fluorescent protein from jellyfish Aequorea Victoria, which has been widely used as reporter for decades. GFP is fused at C terminal with OmpA so that GFP may be displayed on the surface of VnDX.
Usage
Here, we use No part name specified with partinfo tag. to construct the expression system, which may achieve surface display on VnDX.
Characterization
1. Identification
The surface display system for Vibrio natriegens has never been reported before, so the performance of the surface display system of OmpA needs to be verified preferentially. Surface display system bricks (BBa_K3739021, Fig. 1A) were assembled into plasmids, respectively, transformed into Vibrio natriegens through electroporation. After being verified by agarose gel electrophoresis, the plasmids have been transformed into Vibrio natriegens successfully (Fig. 1B).
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- Fig. 1. Gene circuit and agarose gel electrophoresis. (A) Gene circuit of OmpA-GFP (BBa_K373902). (B) Target bands of OmpA-GFP (black arrow, 1900 bp).
2. Proof of the expression
After successful construction and transformation, the experiment was carried out to verify whether the surface display system was expressed and located on the membrane of Vibrio natriegens. Thus, the total membrane proteins were extracted to verify the expression of the surface display system. However, the complicated component of the membrane proteins seriously hinders the analysis of SDS-PAGE (Fig. 2).
- Fig. 2. SDS-PAGE analysis of OmpA-GFP by coomassie blue staining. Target bands of OmpA-GFP (red box, 57.0 kDa).
Western blot analysis was performed, in which the GFP in fusion protein serves as the antigen, and the experimental results are shown in Figure 3. The targeted band was observed in the precalculated position in the western blot results, suggesting that the OmpA-GFP fusion protein was successfully anchored on the surface of Vibrio natriegens.
- Fig. 3. Western Blot analysis of OmpA-GFP. Target bands of OmpA-GFP (white arrow, 57.0 kDa).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 430
Illegal SapI.rc site found at 856
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