Part:BBa_K5136048

inpnc-linker-mt3-linker-mt2a

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. This basic part (BBa_K5136048) which codes the fuesed protein INPNC-linker-MT3-linker-MT2A was constructed and then used for the construction of the composite part (BBa_K5136226) .

Agarose gel electrophoresis (AGE)

I0500 promoter was employed to start the expression of INPNC-linker-MT3-linker-MT2A (BBa_K5136048) in E. coli DH10β. The basic part (BBa_K5136048) is a component of the composite part (BBa_K5136226). The composite part  (BBa_K5136226) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into  E. coli DH10β.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)

Adsorption of Cr₂O₇^2-

After being cultivated and induced by 0.2% L-arabinose at 37 °C for 4 hours ,the engineered bacteria were cultured in M9 medium containing K₂Cr₂O₇ (5 mg/L) for 24 hours. A bit of the supernatant of the bacterial cultures was collected at 0 h and 24 h, and was pre-treated with 1, 5-Diphenyl carbazide (DPC), then the residual amount of Cr₂O₇^2- in the supernatant was determined by measuring the RGB value, which can be calculated based on a standard curve of Cr₂O₇^2- in figure 2 (please see Result for details ).
As shown in Figure 3, after 24 hours, the Cr₂O₇^2- in the culture medium was obviously lower than the initial value. It indicateed that the engineered bacteria could successfully reducethe heavy metal ion Cr₂O₇^2- in wastewater.

Figure 2 Standard curve ofK₂Cr₂O₇(R²=0.987）
Figure 3 Concentration of Cr₂O₇^2- in the culture medium after 0 h and 24 h

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

  Illegal BamHI site found at 330
  Illegal BamHI site found at 975
  Illegal BamHI site found at 1227
  
• 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]