Part:BBa_K4390012

Danio rerio Metallothionein

Usage and Biology

Metallothionein (MT) is a small protein (around 6-7 kDa) which is rich in cysteine. These thiol group in cysteines provide ability to chelate almost all heavy metal ions including Cd2+, Hg2+, Pb2+ and As3+, but had been shown that has higher binding affinity with Hg2+ (Manceau, A. et al., 2019). The ability of chelating heavy metals provides the metal tolerance for its hosts. For its ability to binding heavy metal strongly, this part can be used to build structure which can capture heavy metal ions in aqueous environment. This MT sequence was obtained from Danio rerio, as an isoform which contains higher cysteine frequency with 23 in 72 amino acids (Vergani, L. et al., 2007). To improve the heavy metal binding affinity, Danio rerio MT was compared with MT from Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, Pseudomonas fluorescens and Saccharomyces cerevisiae for their ability to chelate more heavy metals which lead to higher heavy metal tolerance in BL21(DE3). To express and purify the protein, the sequence was designed as a C part for JUMP assembly (Valenzuela-Ortega M and French C., 2021).

Characterization

Danio rerio MT part is required to be assembled into plasmid pJUMP29-1A(lacZ) along with BBa_K4390017 and BBa_K4390016. To confirm the assembly was success, we performed blue-white colony screening and colony PCR. The transformed cells were plate on Kanamycin and X-gal plates. Since pJUMP29-1A(lacZ) contains lacZ as a cloning receptor, the beta-galactosidase encoded by lacZ will cleave X-gal and forming a molecule which dimerizes and turns the colony blue when assembly is failed. It is possible that the lacZ in pJUMP29-1A(lacZ) was cut out and the non-complementary sticky ends were annealled by T4 ligase. Therefore we picked up white colonies and performed colony PCR to ensure that the assembly was correct (Figure 1).

Figure 1. Colony PCR of Danio rerio MT using PS1 and PS2 as primers. The 1 kb ladder (left) and colony PCR products (right) was running through a electrophresis gel to determine the molecular weight of assembled plasmid.

Result

Docking simulation

Non-designed Danio rerio MT sequence was taken from NCBI and the Alphafold structures shown were predicted (Figure 4). These structures were docked to Ag+ using AutoDock 4.2 such that the structures were hydrated and energy minimised while allowing gamma sulphurs on the sidechains of cysteines to form coordinate covalent bonds with the metal ligand (Figure 4).The energy minimisation was done after each ligand was docked. MTs contain many cysteines however each cysteine does not carry the same binding affinity for the ligand. This was accounted for using a pass/fail metric where the passed cysteine had negative Gibbs free energy thus making the binding spontaneous. As result, there were 4 Ag+ docked with Gibbs free energy per ion of -0.145 kcal/mol. This data was compared with Mytilus edulis, Mytilus galloprovincialis, Callinectes sapidus, Pseudomonas fluorescens and Saccharomyces cerevisiae. With the same number of cysteine, Danio rerio MT and Mytilus edulis MT Ag+ binding affinity are similar.

Sequence and Features

Assembly Compatibility: