Part:BBa_K5291036

pAB1-pS-PEBP-GFP

This part is design to anchor the PEBP-GFP fusion protein to the membrane of bacteria. bacteria with PE-binding peptide (PEBP) are expected to have a stronger PE plastic binding capacity. And GFP is used to observe the bacteria on the PE surface to detect the binding ability of the engineering bacteria.

Usage and Biology

In degradation module we constructed pAB1-PEBP-GFP to verify the adsorption capacity of PEBP to PE by fluorescence microscopy.

Fig.1 The map of plasmid pAB1-pS-PEBP-GFP.

The bands of PEBP-GFP from PCR were identical to the theoretical lengths estimated by the designed primer locations, which could demonstrate that we successfully amplified our target fragments.The band was identical to the expected length of 2108bp.

Fig.2 The PCR result of PEBP-GFP fragment.

We transferred the constructed plasmid into Escherichia coli DH5α strain and conducted colony PCR. After obtained the correct result we amplified and extracted the constructed plasmids. Then we transferred these plasmids into Pseudomonas aeruginosa PAO1 strain and obtained correct colony PCR results, indicating that we successfully constructed strain containing the plasmid.

Fig.3 P.aeryginosa PAO1 colony PCR results of PEBP-GFP.

PEBP-GFP proteins was successfully expressed in the strains. SDS-PAGE was performed and the following results were obtained.The plasmid pAB1-pS-PEBP-GFP is expected to express a large fusion protein PEBP-GFP protein with a molecular weight of 67.73kDa. In the figure, it can be seen that compared with the control group, the experimental group had multiple marker bands in the middle of the marker bands slightly smaller than 75kDa and 60kDa, suggesting that the PEBP-GFP protein was successfully expressed.

Fig.4 The SDS-PAGE result of PEBP-GFP.

The PEBP-GFP protein was extracted and purified. After being immersed in PBS and cleaned twice by the vortex shaker, the microplastics incubated with PEBP-GFP still generally showed obvious fluorescence. The microplastics incubated with GFP can see weak fluorescence of GFP residue after intense exposure, while the blank group can not see any fluorescence under the fluorescence microscope. It can be concluded that PEBP has strong binding ability with microplastics.

Fig.5 From left to right are images of microplastics with 500mm diameter incubated with GFP with PE binding peptide, with GFP protein alone, and a blank control.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1033
  Illegal PstI site found at 110
  Illegal PstI site found at 1043
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1033
  Illegal PstI site found at 110
  Illegal PstI site found at 1043
  Illegal NotI site found at 247
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1033
  Illegal BamHI site found at 996
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1033
  Illegal PstI site found at 110
  Illegal PstI site found at 1043
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1033
  Illegal PstI site found at 110
  Illegal PstI site found at 1043
  Illegal NgoMIV site found at 1351
  Illegal NgoMIV site found at 1720
  Illegal AgeI site found at 394
  
• 1000
  COMPATIBLE WITH RFC[1000]