Part:BBa_K5136035

inpnc-His tag-SpyCatcher

Biology

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, the basic part (BBa_K5136035) which codes the fused protein INPNC-his tag-SpyCatcher was constructed and then used for the construction of the composite part (BBa_K5136224) .

Characterization

Agarose gel electrophoresis (AGE)

I0500 promoter was employed to start the expression of INPNC-histag-spycatcher (BBa_K5136035) in E. coli BL21 (DE3). The basic part (BBa_K5136035) is a component of the composite part (BBa_K5136224). 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 around 3000 bp. (Fig. 1).

Fig.1 Colony PCR 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. After induced with 0.2%(w/v) arabinose for 12 hours, the E. coli BL21(DE3) culture solution (express INPNC-his tag-Spycatcher) was incubated with hisTag-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 can be clearly seen from the Fig. 2 that the precipitation fluorescence intensity of the experimental group transferred to INPNC-histag-SpyCatcher_pSB1C3 was significantly higher than that of the control group transferred to I0500_pSB1C3. In addition, we placed the precipitation containing the control group and the experimental group under blue light. We can clearly see that E. coli BL21(DE3) transferred to INPNC-histag-SpyCatcher_PSB1C3 emits green fluorescence, while E. coli BL21(DE3) transferred to I0500_pSB1C3 does not emit green fluorescence (Fig.3). These indicated the combination between HisTag-SpyTag-GFP and the INPNC-HisTag-SpyCatcher displayed in the surface of E. coli BL21(DE3), demonstrating that INPNC has excellent capacity to anchor target proteins on the cell membrane.
<ceter>Fig.2 Fluorescence intensity of precipitation after 12 hours</center>

Fig.3 Samples placed under blue light after 12h

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 NotI site found at 481
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 330
  Illegal XhoI site found at 1089
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 429
  
• 1000
  COMPATIBLE WITH RFC[1000]