Part:BBa_K3739052

J23100-B0030-OmpA-LC1KR-2-GFP-B0010

We anchor LCI-KR2 protein onto membranes through OmpA to stick the engineered bacteria on the polypropylene. We use BBa_K3739052 to construct the expression system and anchor LCI-KR2 on the surface of VnDX.

Biology

OmpA is an anchor protein from E. coli, also find in Vibrio Species, which can anchor its passenger protein to the cell membrane. It has been widely used in cell-surface display. LCI-KR2, A mutant of a peptide called LCI from Bacillus subtilis, can sticks to polypropylene strongly. LCI-KR2 is fused at C terminal with Lpp-OmpA so that LCI-KR2 can be displayed on the surface of VnDX.

Characterization

1. Identification

We attempt to display the plastic-binding protein on the surface of Vibrio natriegens through OmpA, which could endow the ability of plastic-binding to Vibrio natriegens. The gene of OmpA-LCI KR-2-GFP (Fig. 4A, BBa_K3739025) was assembled into the plasmid backbone and 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-LC1KR-2-GFP-B0010 (BBa_K3739025). (B) Target bands of J23100-B0030-OmpA-LC1KR-2-GFP-B0010 (black arrow, 2100 bp).

2. Proof of the expression

After successful construction and transformation, the experiment was carried out to verify whether the OmpA-LCI KR-2-GFP was expressed and located on the membrane of Vibrio natriegens. Thus, the total membrane proteins were extracted to verify the expression of the OmpA-LCI KR-2-GFP. However, the complicated component of the membrane proteins also seriously hinders the analysis of SDS-PAGE (Fig. 2).

Fig. 2. SDS-PAGE analysis of J23100-B0030-OmpA-LC1KR-2-GFP-B0010 by coomassie blue staining. Target bands of OmpA-LC1KR-2-GFP (red box, 61.7 kDa).

The 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 plastic-binding protein (OmpA-LCIKR-2-GFP) was successfully anchored on the surface of Vibrio natriegens.

Fig. 3. Western Blot analysis of J23100-B0030-OmpA-LC1KR-2-GFP-B0010. Target bands of OmpA-LCIKR-2-GFP (white arrow, 61.7 kDa).

3. Verify the plastic binding ability of Vibrio natriegens with plastic-binding proteins on their surface

After successfully displaying the plastic binding protein on the membrane, an experiment was designed to investigate the biding ability of Vibrio natriegens to the plastic microsphere. Firstly, the Vibrio natriegens was cultivated until its OD₆₀₀ reached 0.6, in which OmpA-LCIKR-2-GFP was the experimental group and OmpA-GFP was the control group. Secondly, chloramphenicol was added to the culture solution to inhibit the growth, in which the working concentration was 12.5 μg/mL. Thirdly, after the OD₆₀₀ maintained a stable value, a plastic microsphere was added to the culture solution (0.5 g/mL). Take samples every 5 minutes and measure the OD600 value. As shown in Fig. 4, compared with that in the group of OmpA-GFP (none plastic-binding), the OD₆₀₀ of the OmpA-LCIKR-2-GFP (plastic-binding) decreased markedly. The significant difference demonstrated that the plastic-binding protein (LCIKR-2) displayed in the membrane endow the Vibrio natriegens with the excellent performance of plastic-binding.

Fig. 4. Binding ability of engineered Vibrio natriegens to plastic. (A) The effect from adding plastic microsphere on the OD<sbumit 600> in experiment group (OmpA-LCIKR-2-GFP display on the surface of Vibrio natriegens) and control group (OmpA-GFP display on the surface of Vibrio natriegens). (B) Time course of ∆OD₆₀₀. ∆OD₆₀₀: The value of OD₆₀₀ in each time minus the value of OD₆₀₀ at the time of 0. **** = p < 0.0001.

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 1109