iDEC 2022 | Edinburgh-UHAS_Ghana
Random mutagenesis and silver nitrate (AgNO3) based negative selection of different metallothioneins (MTs) exploring the potential for functional MT directed evolution.
Heavy-metal water pollution is a major public health and biodiversity crisis. Metallothioneins (MT) are cysteine-rich proteins involved in metal-homeostasis and heavy-metal bioaccumulation. The Edinburgh-UHAS_Ghana iDEC team is exploring the potential of MT directed evolution as a mechanism for increasing heavy-metal bioaccumulation. A silver nitrate based negative selection and mutant screening method has been developed to select MTs with higher metal binding capacity. Mutant libraries were created using error-prone PCR for four MTs of interest. A single mutant and its wild-type variant were expressed in BL21(DE3) cells and inputted into the negative selection method. Results indicated that MT expression increases the silver resistance. MTs from six species of interest were structurally modelled via Alphafold and docking simulations using silver as the ligand were performed. Conservation scores from multiple sequence alignments were used to propose functional mutation sites for all MTs of interest and site-specific mutagenic primers were made for the same.