Part:BBa_K5136226

I0500-B0034-inpnc-linker-mt3-linker-mt2a-B0015

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).

MT3 and MT2A

The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can bind to 6-9 heavy metal ions. The MTs are expressed as intracellular protein and are primarily responsible for metal regulation in cells of living organisms. General MTs can widely non-covalently bind divalent heavy metal ions, such as Zn²⁺, N²⁺, Pb²⁺, Hg²⁺, Cd²⁺, as well as As³⁺, but their effectiveness in treating Cr₂O₇^2- is not satisfactory. MT2a and MT3 are metallothioneins(MTs) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr₂O₇^2-. And MT3 has a better adsorption effect on ordinary metal ions (2).

Usage and Biology

After deinking, heavy metal ions in the ink will be released into the wastewater. Thus, this wastewater needs harmless treatmentremove before discharging. Hence, we use MTs to treat wastewater to remove heavy metals. And, in order to increase the contact area of MTsand metal ions to improve the adsorption efficiency, we introduce INPNC for surface display of MTs. Thus, this composite part BBa_K5136226 was constructed to express the fuesed protein INPNC-linker-MT3-linker-MT2A which anchored on the surface of bacteria.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136226) 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 (4464 bp) can be observed at the position around 5000bp (Figure 1).

Figure 1 Colony PCR of BBa_K5136226_pSB1C3 in E. coli BL21(DE3)

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. A. A. Uçkun, M. Uçkun, S. Akkurt, Efficiency of Escherichia coli Jm109 and genetical engineering strains (E. coli MT2, E. coli MT3) in cadmium removal from aqueous solutions. Environ. Technol. Innovation 24, 12 (2021).

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 BamHI site found at 2211
  Illegal BamHI site found at 2466
  
• 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