Part:BBa_K5136037

mt3

Biology

MT3

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⁺,Ni²⁺, Pb²⁺, Hg²⁺, Cd²⁺, as well as As³⁺. MT3 has a excellent adsorption effect on ordinary metal ions. (1).

Usage and Design

To absorb Cd²⁺ in waste water, we introduced MntA membrane protein in E. coli, which translocates external Cd²⁺ into the cell. To increase the strain's tolerance to Cd²⁺, we produce intracellular MTs in engineered bacteria, mitigating the cell damage of Cd²⁺. This basic part (BBa_K5136037) which codes the MT3 was constructed and then used for the construction of the composite part (BBa_K5136230) .

Characterization

Agarose gel electrophoresis (AGE)

I0500 promoter was employed to start the expression of MT3 (BBa_K5136037) in E. coli BL21(DE3). The basic part (BBa_K5136037) is a component of the composite part (BBa_K5136230). The composite part  (BBa_K5136230) 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 (2106 bp) can be observed at the position around 2000bp (Figure 1).

Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in E. coli BL21(DE3)

Reference

1. 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 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]