Part:BBa_K3394000

Coding Sequence of Endo5a cellulase (for E. coli)

This part consists the coding region of Endo5A, an endo-1,4-beta-glucanase. Endo5a breaks down cellulose by cutting internal 1-4 beta glucose linkages in the cellulose chain.

Figure 1: Cellulose chain. 1-4 beta glucose linkage is highlighted

The complete cellulose hydrolysis reaction involves this enzyme along with several other enzymes: endo-1,4-beta-glucanases, exo-1,4-beta-glucanases (or cellobiohydrolases) and beta-glucosidases.

Endo5a is a relatively small protein with an optimal temperature of 50 Celsius degrees and an optimal pH range of 6-7. The NCBI Genbank number for Endo5a is HQ657203.1 (DNA), AEB00655.1 (amino acid).

We learned about the cellulase from a past iGEM team – the British Columbia 2016 team which fused it to the surface layer of Caulobacter crescentus (BBa_K2139001). In our project, we optimized the sequence for use in Escherichia coli, and it was synthesized by IDT for us. Furthermore, Endo5a was expressed with an N-terminal histidine tag (6-histidine tag) instead of the 17 first amino acids that served as a signal peptide (51 nucleotides).

Endo5A was isolated from the bacterial flora found in the gut of a cotton bollworm (Helicoverpa armigera) which secretes a variety of plant-hydrolyzing enzymes.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 898
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 898
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 898
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 898
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 898
  
• 1000
  COMPATIBLE WITH RFC[1000]