Part:BBa_K3739102

J23100-B0034-INPNC-his-SpyCatcher-B0010

The SpyCatcher protein is anchored on the membranes through INPNC. This design is meant to polish the demonstration on the anchoring effect of INPNC.
INPNCIt is a membrane protein commonly used for the surface display system of E. coli, through which the SpyCatcher protein could anchor on the membranes. At the same time, SpyTag in the environment could bind to SpyCatcher by forming an isopeptide bond (Fig. 1). Based on this design, we aim at improving the usability and success of targeted protein anchoring in the surface display system. A new composite part (BBa_K3739102) was constructed to express the protein of INPNC-HisTag-SpyCatcher.

Fig. 1. Schematic diagram of bonding process between INPNC-HisTag-SpyCatcher and HisTag-SpyTag-GFP.

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. SpyTag is a peptide fragment, which could steadily bind to SpyCatcher by forming an isopeptide bond.

Usage

Here, we construct the genetic circuit to express INPNC-SpyCatcher on the surface of E. coli, and characteristic the effect. The composite part BBa_K3739102 constructed was introduced into the backbone plasmid through standard assembly and transformed into E. coli BL21 (DE3). The positive clones were selected, verified and cultivated.

Characterization

====1. Identification====
PCR was employed to verify the correctness of the plasmid constructed. As shown in Fig. 2, the targeted band (1564 bp) could be observed at the position around 1500 bp of the marker band.

Fig. 2. DNA gel electrophoresis of PCR products of INPNC-SpyCatcher (Xbal I & Pst I sites).
====2. Proof of the expression====
J23100 promoter was employed to start the expression of INPNC-SpyCatcher (BBa_K3739102) in E. coli. After selected and verified, the positive colony was cultivated overnight to express the fusion membrane protein of INPNC-SpyCatcher.

====3. Bonding ability to SpyTag====
1 mL E. coli BL21(DE3) culture solution, which has express fusion membrane protein of INPNC-Spycatcher, was used for the following experiment. Another engineered E. coli BL21(DE3) with HisTag-Spytag-GFP was also cultured, and harvest the bacteria in sediment after being centrifuged with the condition of 8000 g, 10 min, and 4°C. After broken and centrifuged, the supernatant solution with HisTag-Spytag-GFP was obtained and incubated with the E. coli BL21(DE3) culture solution (express INPNC-Spycatcher) at 37 °C in the shaker. Samples were taken every two hours. After centrifugation, the fluorescence of 50 μL supernatant of each sample was measured ( λ_ex = 475 nm, λ_em = 545 nm), in which the bacteria incubated with PBS solution was set as control. At the same time, the fluorescence confocal microscope has also been employed to verify the bonding of SpyTag to INPNC-SpyCatcher (Fig. 3). After incubation, the decrease of fluorescence intensity of supernatant indicated the binding of HisTag-SpyTag-GFP to the INPNC-HisTag-SpyCatcher displayed in the surface of E. coli BL21(DE3), while the fluorescence intensity in the control group changes slightly (Fig. 4).

Fig. 3. The bonding of INPNC-HisTag-SpyCatcher to SpyTag observed from fluorescence confocal microscope.

Fig. 4. Time course of fluorescence changes of the supernatant.

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 133
  Illegal AgeI site found at 884
  
• 1000
  COMPATIBLE WITH RFC[1000]