Part:BBa_K4011008

CBM3-NT2RepCT-CBM3

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_K4011000 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, CBM3s are expressed as a domain of a protein whose functions require being bound to cellulose, such as cellulase. The structure of CBM3 is displayed in the Characterization section (Protein Data Bank accession: 1NBC).

CBM3 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 CBM3 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

CBM3 is derived from Ruminiclostridium thermocellum. NT2RepCT is an artificial spider silk fibroin (Visit BBa_K3264000 for more).

Design Considerations

1. All codons were optimized for E. coli based on E. coli codon bias

2. Flexible linker GSGGGS used between first CBM3 and NT2RepCT, flexible linker GGGGS used between NT2RepCT and second CBM3.

3. 6x histag (MGHHHHHHM) added to C-terminal to allow for Ni-NTA purification.

Characterization

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 91
  Illegal AgeI site found at 301
  Illegal AgeI site found at 1609
  Illegal AgeI site found at 1819
  
• 1000
  COMPATIBLE WITH RFC[1000]