Part:BBa_K5136224

I0500-B0034-inpnc-His tag-SpyCatcher-B0015

INPNC

Ice nucleoprotein (INP), an outer membrane protein from Pseudomonas syringae, has been used as a surface anchor in many researches. The truncated version of INP, namely INPNC which contains only the N- and C-terminal portion of INP, has excellent capacity to anchor target proteins on the cell membrane (1).

SpyCatcher

SpyCatcher is a engineered split fragment of the fibronectin-binding protein (FbaB) in Streptococcus pyogenes, containing 116 residues. SpyTag is a peptide tag of 13 residues developed by previous researchers. SpyCatcher could steadily bind to SpyTag by forming an isopeptide bond (2).

Usage

We aim to displaying metallothioneins MT2A and MT3 on the surface of E. coli by INPNC, which could improve the adsorption rate of heavy metal ions for the harmless treatment of deinking wastewater. However, it is tricky to verify whether a protein has been successfully displayed on the surface of bacteria. So, we introduce the Spy system. In our project, SpyCatcher is fused with INPNC and SpyTag is fused with GFP. Then, engineered bacteria which express the fused protein INPNC-His tag-SpyCatcher are treated with SpyTag-GFP. By monitoring the fluorescence intensity of bacteria, we can verify if INPNC could anchor target proteins on cell surface. Here, we constructed the composite part (BBa_K5136224) to express INPNC-His tag-SpyCatcher.

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136224) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli BL21 (DE3). The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2974 bp) can be observed at the position around 3000 bp (Figure 1).

a.

b.

Figure 1 Constructing INPNC-His tag-SpyCatcher fused protein. a. the expression gene circuits for INPNC displaying SpyCatcher on the surface of E. coli BL21(DE3). b. DNA gel electrophoresis of the colony PCR products of BBa_K5136224_pSB1C3 in E. coli BL21(DE3)

Fluorescence Intensity Determination

In order to verify the surface display effect of INPNC, plasmid pSB1C3 bearing composite part BBa_K5136224 was transformed into E. coli BL21(DE3) for subsequent verification experiments, and E. coli BL21(DE3) carrying the I0500_pSB1C3 vector was set as the control group. After induced with 0.2%(w/v) arabinose for 12 hours, the culture solution of the experimental group(expressing INPNC-His tag-Spycatcher) and the culture solution of the control group were incubated with His tag-SpyTag-GFP at 37 °C in the shaker. Cultures were taken after 12 hours, then were centrifuged to get the precipitate. The fluorescence intensity of the samples were measured. It was clearly seen from the Figure 2 that the fluorescence intensity of the experimental group was significantly higher than the fluorescence intensity of the control group. In addition, we placed the precipitation containing the control group and the experimental group under blue light. We clearly saw the green fluorescence of the experimental group excited by blue light, while the control group was contrary (Figure 3). This fact indicated the combination between His tag-SpyTag-GFP and the INPNC-His tag-SpyCatcher displayed on the surface of E. coli BL21(DE3), demonstrating that INPNC has excellent capacity to anchor target proteins on the cell membrane.

Figure 2 Fluorescence intensity of samples after culturing 12 hours.

Figure 3 Samples placed under blue light after culturing 12 hours.

Reference

1. M. Shimazu, A. Mulchandani, W. Chen, Cell surface display of organophosphorus hydrolase using ice nucleation protein. Biotechnology Progress 17, 76-80 (2001).
2. B. Zakeri et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NotI site found at 1717
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 1566
  Illegal XhoI site found at 2325
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1308
  Illegal NgoMIV site found at 1641
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1665
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961