Difference between revisions of "Part:BBa K5136228"

Revision as of 09:01, 1 October 2024

I0500-B0034-gst-linker-mt2a-B0015

Biology

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 26KD 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).

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⁺, Ni²⁺, Pb²⁺, Hg²⁺, Cd²⁺, as well as As³⁺, but their effectiveness in treating Cr₂O₇^2- is not satisfactory. MT2a is a metallothioneins(MT) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr₂O₇^2- (2).

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. Thus, this composite part BBa_K5136228 was constructed to express fused protein GST-linker-MT2A.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136228) 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 (2560 bp) can be observed between 2000 bp and 3000 bp (Figure 1).