Part:BBa_K3332063

J23100-RBS-GFP-AIDA-terminator

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

Biology

        AIDA as bacterial display system is a kind of autotransporter family which can anchor to the membrane. It is from E. coli. 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 AIDA so that EGFP can be displayed on the surface of E. coli.

Usage

        Here, we use BBa_K880005 to construct the expression system and obtain the composite part BBa_K3332063, which may achieve surface display of EGFP on our engineering bacteria.

Fig 1. Gene circuit of EGFP-AIDA.

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 EGFP-AIDA-pSB1C3.

2. 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 3, we could find that both the bacteria and bacteria periciptation after ultrasonication 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-AIDA have fluorescence signal always only on the 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 3. (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); (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.

3. Microscopic observation

        we used fluorescence microscopy to see if AIDA worked and the experimental result were shown on Figure6. 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_K3332063 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 AIDA.

Fig 4. 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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2055
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 705