Bacterial metallothionein SmtA (BBa_K4968001), originating from Synechococcus PCC 7942, is a protein belonging to the metallothionein family. It can sense and bind specific heavy metal ions such as cadmium, lead, zinc, etc., thereby assisting in regulating the cell's tolerance and metabolism of these toxic metals. In our project, we utilized the metallothionein SmtA as the protein that mainly adsorbs heavy metal ions. In addition, we optimized the codons of SmtA before placing them on the plasmid.
Carbohydrate Binding Modules (CBM) (BBa_K4968002) are components of several enzymes, and their primary function is to bind to specific carbohydrates. In our project, we have chosen a family 2 CBM from endoglucanase A of C.fimi. In addition, we optimized the codons of the CBMs before placing them on plasmids to improve expression efficiency in strains.
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. Before placing it on the plasmid, we optimized the codon sequence of sfGFP to improve its transcription efficiency within the strain.
Figure 1: This picture shows the conformation of the sfGFP and CBM binding to the microcrystalline cellulose.
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. In our project, we optimized the Condon codes before putting them on the plasmid.
The CsgA protein is the kind of amyloid structure on the outer membrane of the E.coli. It can be assembled in the cell body in the presence of the CsgB which could also anchor the CsgA protein on the outer membrane after secreting. Meanwhile, without CsgB, the CsgA protein could also self-assemble extracellularly. According to Courchesne et al.(2016), CsgA could be separated and form independent curli nanofibers. In our project, we use the CsgA as the backbone of our target protein AG4 (BBa_K4968005). We optimized the Condon codes before putting the CsgA sequence on the plasmid.
The AG4 peptides are the kind of artificially synthesized 12-mer peptides that could specifically attract silver ions. The size of the protein that could adsorb silver ions is limited because the maximum size of the fusion protein that could allow it to go through the protein is 59 amino acids. In our project, we took advantage of the relatively small mass of the size of AG4, combining it with CsgA (BBa_K4968004) to get CsgA-AG4 (BBa_K4968008) fusion protein. We optimized the Condon codes before putting the AG4 sequence on the plasmid.
pUC-GW-Kan R (BBa_K4968012) is a commonly used plasmid cloning vector in E. coli. The vector length is 2,626 bp. GW represents "Gateway" technology, which is commonly used for efficient gene cloning and expression. Gateway technology allows researchers to easily move gene fragments into different plasmids, achieving targeted cloning and expression of target genes. The plasmid contains the kanamycin antibiotic resistance gene for screening cells carrying this plasmid when cultivating E. coli.
| Type | Part Number | Name | Length | Description |
|---|---|---|---|---|
| Basic | MSmtA4 | 168 bp | MSmtA4, a mutant protein derived from SmtA (BBa_K4968001) with four mutated sites and codon optimization, effectively enhances the adsorption capacity of heavy metals | |
| Basic | Optimized SmtA | 168 bp | SmtA, a protein belonging to the metallothionein family, and it has the ability to adsorb heavy metals and chelate these heavy metals in an inactive form. | |
| Basic | Optimized CBM | 315 bp | Carbohydrate Binding Modules (CBMs) are components of several enzymes, and their primary function is to bind to specific carbohydrates. | |
| Basic | Optimized sfGFP | 711 bp | sfGFP, a fluorescent labeling tool, does not lead to misfolding when fused with other proteins, thereby enhancing the stability of the fused protein. | |
| Baisc | Optimized CsgA | 453 bp | CsgA, a gene has remarkable ability to self-assemble into curli fiber amyloid nanofibers, optimized through codon optimization. | |
| Basic | Optimized AG4 | 39 bp | AG4, a synthetic silver-binding dodecapeptide, optimized through codon optimization. | |
| Basic | RecomSwapNeo R/Kan R | 1528 bp | The improved part of Low to medium copy Lambda Red recombineering compatible plasmid (BBa_K592202). As a Vector for Lambda Red Homologous Recombination. | |
| Basic | pUC_GW_Kan R | 2626 bp | As a Plasmid backbone for the part CBM-sfGFP-pUC_GW_Kan R-SUMO (BBa_K4968017). | |
| Tag | Optimized SUMO tag | 294 bp | Small ubiquitin-like modifiers (SUMOs) are widely used as tags for protein expression and purification. |
Courchesne, N.M.D., et al. (2017) ‘Scalable Production of Genetically Engineered Nanofibrous Macroscopic Materials via Filtration’ American Chemical Society 3, 5, 733–741. Available at: https://doi.org/10.1021/acsbiomaterials.6b00437