Part:BBa_K5136041

gst-linker-crs5

GST tag

Glutathione-S-transferase (GST) tag is a peptide tag derived from Schistosoma japonicum. GST tag has a large relative molecular mass of about 26 KDa and is often inserted at the N-terminus of target proteins. It facilitates the separation of target proteins from cell extracts by its affinity for glutathione In addition, most of these fusion proteins are stable and water-soluble.(1).

CRS5

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⁺. CRS5 is a metallothionein from S. cerevisiae. It is mainly responsible for copper ion homeostasis and detoxification in yeast cells. (1).

Usage and Design

We decided to use MTs to treat wastewaters. In order to increase the stability of MTs, we added a GST tag to its N-terminal. This basic part (BBa_K5136041) which codes the fused protein GST-linker-CRS5 was constructed and then used for the construction of the composite part (BBa_K5136229) .

Agarose gel electrophoresis (AGE)

I0500 promoter was employed to start the expression of GST-linker-CRS5 (BBa_K5136041) in E. coli DH10β. The basic part (BBa_K5136041) is a component of the composite part (BBa_K5136229). The composite part (BBa_K5136229) 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 (2584 bp) can be observed at the position between 2000 bp and 3000 bp(Figure 1).

Figure 1 Colony PCR of BBa_K5136229_pSB1C3 in E. coli DH10β

Reference

1.V. C. Culotta, W. R. Howard, X. F. Liu, CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J. Biol. Chem. 269, 25295-25302 (1994). 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
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 673
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 85