Part:BBa_K3332064

J23100-RBS-GFP-BrkA-terminator

        This is an anchor proteins onto membranes through BrkA and use GFP to comformation. We use BBa_K880005 and GFP to verify BrkA's function which can anchor proteins onto membranes.

Biology

        BrkA (Bordetella serum-resistance killing protein A) is from Bordetella pertussis. It can anchor its passenger protein to the cell membrane and has been widely used in cell-surface display.

        EGFP is green fluorescent protein from jellyfish Aequorea Victoria, which has been widely used as reporter for decades. EGFP is fused at C terminal with BrkA so that EGFP can be displayed on the surface of E. coli.

Usage

        Here, we used BBa_K880005 to construct the expression system. We obtained the composite part BBa_K3332064 and transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression and activity. The positive clones were cultivated.

Fig 1. Gene circuit of EGFP-BrkA.

Characterization

1.Identification

        After receiving the synthesized DNA, PCR was done to certify that the plasmid was correct, and the experimental results were shown in figure 2.

Fig 2. DNA gel electrophoresis of PCR products of EEGFP-BrkA-pSB1C3.

2.The proof of expression

        We used BBa_J23100 promoter to highly express EGFP-BrkA in E. coli in our composite part BBa_K3332064. In order to verify whether our recombinant plasmid works, we use membrane protein extraction kit to get membrane protein.

        Then, our target bands are observed through SDS-PAGE and the experimental results are shown in figure 3. Notably, in EGFP-BrkA lane we could see our target protein band which do not show up in negative controls and we could not see EGFP protein band which shows up in EGFP.

Fig 3. SDS-PAGE of membrane protein extraction products of EGFP-BrkA-pSB1C3.

3.Quantitative detection of fluorescence

        In order to verify whether EGFP was anchored to the cell membrane, we detected the fluorescence of bacteria washed by PBS Buffer and bacteria periciptation after ultrasonication.

        By comparing the fluorescence of samples on the photos on Figure 4, we could find that both the bacteria and broken thallus of E. coli with J23100-RBS (BBa_K880005) have no fluorescence signal. Both the bacteria and bacteria periciptation after ultrasonication of E. coli with EGFP has strong fluorescence signal. Notably, after ultrasonication the fluorescence signal dispersed to both the supernatant and the precipitation, which means the EGFP protein normally express on the intracellular. However, samples of EGFP-BrkA have fluorescence signal always only on the thallus precipitation, which suggests that our anchor protein successfully display EGFP on the surface of E. coli.

        We further quantitatively detected fluorescence of the resuspension and supernatant of broken. And we could find ratios of fluorescence of the resuspension and supernatant have obvious difference between experimental groups. The results were shown on figure 4.

Fig 4. (a) From left to right, bacteria of E. coli with EGFP-AIDA#3, EGFP and Blank (J23100-RBS (BBa_K880005)); (b) From left to right, bacteria of E. coli with EGFP-AIDA#4, EGFP and Blank (J23100-RBS (BBa_K880005)); (c) From left to right, bacteria of E. coli with EGFP-BrkA, EGFP and Blank (J23100-RBS (BBa_K880005)); (d) From left to right, broken thallus of E. coli with Blank (J23100-RBS (BBa_K880005)), EGFP-AIDA#3 and EGFP; (e) From left to right, broken thallus of E. coli with Blank (J23100-RBS (BBa_K880005)), EGFP-AIDA#4 and EGFP; (f) From left to right, broken thallus of E. coli with Blank (J23100-RBS BBa_K880005)), EGFP-BrkA and EGFP. (g) Quantitative detection of fluorescence of the resuspension and supernatant of broken bacteria.

4.Microscopic observation

        we used fluorescence microscopy to see if BrkA worked and the experimental result were shown in Figure 6. E. coli BL21 (DE3) carrying BBa_E0040 (EGFP) was rod-shaped and the fluorescence was equably distributed in the bacteria. However, the fluorescence of E. coli BL21 (DE3) carrying BBa_K3332064 was observed to be dotted and dispersed on the surface of E. coli. The results proved that EGFP has apparently been anchored to the surface of the E. coli by the function of BrkA.

Fig 5. confocal laser scanning microscope (clsm) imaging

References

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1088
  Illegal NgoMIV site found at 1517
  Illegal NgoMIV site found at 2153
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 705
  Illegal BsaI.rc site found at 2121