Part:BBa_K3739049

J23100-B0030-OmpA-GFP-B0010

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 BBa_3739020 to 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_K3739049, 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). ""

Fig. 1. Gene circuit and agarose gel electrophoresis. (A) Gene circuit of J23100-B0030-OmpA-GFP-B0010 (BBa_K3739049). (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 J23100-B0030-OmpA-GFP-B0010 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 J23100-B0030-OmpA-GFP-B0010. Target bands of OmpA-GFP (white arrow, 57.0 kDa).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 494
  Illegal SapI.rc site found at 920