Part:BBa_K3332061

J23100-RBS-INPNC-GFP-terminator

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

Biology

        Ice nucleoprotein is an anchor protein from Pseudomonas syringae. It can anchor its passenger protein to the cell membrane. N and C terminal of Ice nucleoprotein, which is named after INPNC, can also anchor passenger protein fused with it to the cell membrane.

        EGFP is green fluorescent protein from jellyfish Aequorea Victoria, which has been widely used as reporter for decades. EGFP is fused at N terminal with INPNC 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_K3332061 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 INPNC-EGFP.

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 figure2.

Fig 2. DNA gel electrophoresis of PCR products of INPNC-EGFP-pSB1C3.

2.The proof of expression

        We used J23100 promoter to highly express INPNC-EGFP in E. coli in our composite part BBa_K3332061. 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 2. Notably, in INPNC-EGFP we could see our target protein band which don’t 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 INPNC-EGFP-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 thallus broken by ultrasound.

        By comparing the fluorescence of samples on the photos of Figure5, we could find that both the bacteria and broken thallus of E. coli with J23100 have no fluorescence signal. Both the bacteria and broken thallus 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 broken INPNC-EGFP have most of fluorescence signal 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 figure6.

Fig 4. (a) From left to right, bacteria duplicate samples of E. Coli with Blank(J23100-RBS(BBa_K880005)), INPNC-EGFP and EGFP; (b) From left to right, broken bacteria duplicate samples of E. Coli with Blank (J23100-RBS(BBa_K880005)), INPNC-EGFP and EGFP. (c)Quantitative detection of fluorescence of the resuspension and supernatant of broken.

4.Microscopic observation

        we used fluorescence microscopy to see if INPNC worked and the experimental result were shown on Figure6. E. coli BL21 carrying BBa_E0040 (EGFP) was rod-shaped and the fluorescence was equably distributed in the bacteria. However, the fluorescence of E. coli BL21 carrying BBa_K3332061 was observed to be equably distributed in the bacteria and rarely dispersed on the surface of E. coli. The results proved that EGFP may not be anchored to the surface of the E. coli by the function of INPNC. We need more certification.

Fig 6. 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 466
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1680