Part:BBa_K4011009

CBM2-NT2RepCT-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. CBM3-NT2RepCT-CBM3 is constructed from CBM3 BBa_K4011000 and artificial spider fibroin NT2RepCT BBa_K3264000. This is part in a part collection where we characterize bacterial cellulose modification methods and constructs using CBMs.

The part collection includes: Cellulose binding matrixes 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.

NT2RepCT (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.

The CBM2 connects the 2Rep to our bacterial cellulose membrane (BCM). The 2Rep proteins will also form hydrogen bonds with each another, in the gaps of cellulose fibers in BCM, creating a denser net made of two different materials, thus increasing the physical properties of BCM.

Source

CBM2 is derived from Cellulomonas fimi.. NT2RepCT is an artificial spider silk fibroin (Visit BBa_K3264000 for more).

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’re 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 52
  Illegal AgeI site found at 96
  Illegal AgeI site found at 268
  Illegal AgeI site found at 334
  Illegal AgeI site found at 1444
  Illegal AgeI site found at 1488
  Illegal AgeI site found at 1660
  
• 1000
  COMPATIBLE WITH RFC[1000]