Difference between revisions of "Part:BBa K4011001"

Latest revision as of 05:55, 2 October 2024

CBM2

CBMs are artificial proteins derived from natural proteins with cellulose-binding functions, such as cellulase. There are three types of CBMs, which are CBMs, CBM1, CBM2, and CBM3. CBM1 is the smallest, whilst CBM3 is the biggest. By fusing CBMs to functionalization proteins, we can achieve modification/functionalization of our bacterial cellulose membrane. This is part in a part collection where we characterize bacterial cellulose modification methods and constructs using CBMs.

The part collection includes: Cellulose binding matrix BBa_K4011000 and BBa_K4011001. CBMs fused with spider silk fibroin BBa_K4011008 and BBa_K4011009. Fused proteins capable of expression and secretion in S. cerevisiae BBa_K4011010 and BBa_K4011011.

This part collection can help and inspire other teams we are trying to achieve modification of cellulose membranes using different modification/functionalization proteins.

Usage and Biology

In nature, CBM2s are expressed as a domain of a protein whose functions require being bound to cellulose, such as cellulase. The structure of CBM2 is displayed in the Characterization section (Protein Data Bank accession: 1EXG).

CBM2 fused with spider silk proteins is first done by Mohammadi et al in 2019, where they tested the changes in physical properties on cellulose fibers after mixing with CBM2-spider silk.

Source

CBM2 is derived from Cellulomonas fimi

Characterization

In order to modify BCM’s physical properties, we designed and expressed spider silk fibroins fused with cellulose binding matrixes (CBMs; learn more on our description page) to bind to BCM (Fig. 1). For our project, we experimented with CBM3 from Ruminiclostridium thermocellum (Protein Data Bank (PDB) accession: 1NBC; Fig. 1D) (2) and CBM2 from Cellulomonas fimi (PDB accession: 1EXG; Fig. 1C). For our spider silk protein, we chose to use the synthetic mini spider silk protein NT2RepCT (2Rep; first characterized by GreatBaySZ_2019). 2Rep is water-soluble due to hydrophilic interactions of protein N-terminal and C-terminal. When 2Rep is submerged in a coagulating bath and subjected to a shear force, the repetitive regions will uncoil, form beta-pleated sheet networks and solidify into silk fiber (Fig. 1A).

Figure 1: Structure and design of 2Rep and 2Rep fused with CBMs. A) Schematic representing how individual 2Rep proteins becomes silk fibers. B) Design of CBM fused with 2Rep

For adding CBM3 flanking to 2Rep, we synthesized CBM3-BsaI-CBM3 on a pET28a vector. Primers were then used to add BsaI restriction sites in 2Rep to fuse the respective domains together in the synthesized pET28a vector by Golden Gate assembly (Fig. 2A & 2B). After construction, the plasmids were transformed into E. coli BL21(DE3) for IPTG-inducible expression (Fig. 2C).

Comparing the solubility of CBM2/3 fused with 2Rep

2Rep was shown to possess good water solubility, but we were uncertain whether our modification will change this desirable property. To test the solubility, we cultured the modified strains, induced the expression of the constructs, collected the cells and performed SDS-PAGE on cell lysate (Fig. 2). Expression of both constructs, CBM3-2Rep-CBM3 (72kDa) and CBM2-2Rep-CBM2 (65kDa), are observed. CBM2-2Rep-CBM2 was present in the whole cell sample, but absent in the cell lysate supernatant, indicating poor water solubility. In contrast, CBM3-2Rep-CBM3 was present in both whole cell and supernatant. Therefore, CBM3-2Rep-CBM3 was chosen for the rest of the project for its superiority in water solubility.

Figure 2: CBM-2Rep-CBM construction and expression. A) Schematic representing construction of CBM-2Rep-CBM plasmids using golden gate assembly. B) Gel electrophoresis of CBM-CBM and CBM-2Rep-CBM plasmids. C) Schematic representing transformation of CBM2-2Rep-CBM2 and CBM3-2Rep-CBM3 into E. coli DH5α D) SDS-PAGE analysis of CBM3-2Rep-CBM3 and CBM2-2Rep-CBM2.

Sequence and Features

CONTRIBUTION: LINKS-China 2024 | CBM2-mTurquoise

Authors iGEM LINKS-China 2024, Tong Ding

Carbohydrate-Binding Module 2 (CBM2 BBa_K4011001) is a protein domain found in carbohydrate-active enzymes, particularly those involved in the degradation of cellulose and hemicellulose. CBM2 enhances the ability of these enzymes to bind to their polysaccharide substrates, improving catalytic efficiency. By fusing CBM2 with functional proteins, we can target cellulose-containing and hemicellulose-containing materials for modification or degradation. We selected mTurquoise (BBa_K2722003) as the chromoprotein to be fused with CBM2 to enhance visualization of protein binding ability.

Usage and Biology

In nature, CBM2 is expressed as a domain within cellulases and other glycoside hydrolases that degrade cellulose and hemicellulose, which are major components of plant cell walls. The binding of CBM2 to these polysaccharides increases catalytic efficiency by creating a better fit between the enzyme and substrate surfaces. The structure of CBM2 is documented in the Protein Data Bank (accession: 6F7E). mTurquoise is a chromoprotein that emits bright cyan fluorescence, commonly used in molecular biology applications. This protein serves as a visual marker, enabling researchers to track and confirm the expression of fusion proteins in various experimental setups. By incorporating mTurquoise into our constructs, we can enhance the visualization of protein localization and interactions, thereby improving the overall effectiveness of our assays. CBM2 has applications in biotechnological processes, including biomass conversion, biofuel production, and the recycling of plant materials.

Source

CBM2 is derived from organisms that produce cellulose- and hemicellulose-degrading enzymes, such as certain bacteria and fungi. CBM2-mTurquoise source: This wiki.

Results

We obtained a fusion protein composing of a binding domain protein linked to a fluorescent protein, naming it CBM2-mTurquoise. Figure A shows the results of the SDS-PAGE analysis of the target fusion proteins (BDC). The CBM2-mTurquoise fusion protein was expressed in E. coli BL21 (DE3). CBM2-mTurquoise has a molecular weight of 40 kDa and is successfully expressed. The color of CBM2-mTurquoise suspended in 20mM Tris-HCl under blue light is shown in Figure B, shining in the correct color cyan. The ability of CBM2 to bind with cellulose is assessed, shown in Figure C. The result indicates that CBM2 can successfully bind to cellulose material, as shown by the comparative brightness in color under blue light.