MSmtA4 (BBa_K4968000):

The bacterial metallothionein SmtA (BBa_K4968001) from Synechococcus PCC 7942 is a protein belonging to the metallothionein family. Based on the SmtA protein from Synechococcus PCC 7942, we designed a protein variant called MSmtA4. By mutating three positively charged amino acids (Arg 26, Lys 8, and Lys 22) to Cys, we effectively improved the adsorption capacity of the protein for heavy metal ions, especially cadmium ions, without affecting the structure and function of the protein.

Figure 1: MSmtA docking result. Cd2+ forms ionic bonds with Cys14, Cys54, Cys52 and Cys47. (Docking score: -6.133kcal/mol).

SUMO-SmtA-CBM-sfGFP (BBa_K4968019):

SUMO (Small Ubiquitin-like Modifier) (BBa_K4968007) is a ubiquitin-like protein that is commonly found in eukaryotic organisms. When SUMO is fused to the N-terminus of the target protein, it can improve the folding of the target protein, increase its solubility, and increase protein yield. We use SUMO as a tag to attach to the target fusion protein gene to improve the solubility and yield of the fusion protein to increase the protein yield.

MSmtA4-CBM-sfGFP (BBa_K4968020):

We designed to mutate four sites of SmtA in order to increase the electronegativity on the surface of the fusion protein. The reduced binding energy indicates that the fusion protein can react more easily with heavy metals. Carbohydrate Binding Modules (CBM) (BBa_K4968002) are components of several enzymes, and their primary function is to bind to specific carbohydrates. sfGFP (BBa_K4968003) is a fluorescent marker that does not induce misfolding when fused to other proteins, thereby increasing the stability of the fusion protein. Under daily visible light irradiation, substances expressing sfGFP will show strong green fluorescence. sfGFP can fold and function faster compared to ordinary GFP.

Figure 2: This figure explains the procedure after expressing the fusion protein. The process of adsorbing heavy metal ions.

This fusion protein has several advantages. First, MSmtA4 (BBa_K4968000) plays a crucial role in sensing and regulating heavy metal ions, which significantly enhances the adsorption capacity of the protein for heavy metal ions. In addition, the carbohydrate-binding module (CBM) can be used to modify the surface of cellulose, improving its dispersibility and suspension stability, thus providing potential applications for environmentally friendly materials. Finally, the Superfolder Green Fluorescent Protein (sfGFP) exhibits excellent folding performance and maintains a stable folded state even when fused with other proteins, making it a reliable labeling tool widely used in protein research. This multifunctional fusion protein demonstrates flexibility, versatility, and convenience in various biological research and engineering applications.

SUMO-MSmtA4-CBM-sfGFP (BBa_K4968021):

SUMO (Small Ubiquitin-like Modifier) (BBa_K4968007) is a ubiquitin-like protein commonly found in eukaryotic organisms. When SUMO is attached to the N-terminus of the target protein, it can improve the folding of the target protein, increase its solubility, and increase protein yield. In our project, we optimized the Condon codes before inserting them into the plasmid.

SUMO-MSmtA4-CBM-sfGFP (BBa_K4968021) aims to use MSmtA4 (BBa_K4968000) to adsorb heavy metal ions while improving the solubility and yield of the fusion protein.

RecomSwapNeo R/Kan R (BBa_K4968006):

Considering that we need to knock out the CsgA and CsgB genes in the genomes of BL21(DE3) and MC4100 by ourselves. By searching in NCBI, we designed homologous fragments of 50 bp upstream and downstream of CsgA and CsgB genes. And the kanamycin sequence was referenced from pKD4. On the new plasmid, we designed 50 bp homologous fragments upstream and downstream of the kanamycin gene.