Part:BBa_K3394000

Coding Sequence of Endo5a cellulase (for E. coli)

This part consists on the coding region of Endo5A, an endo-1,4-beta-glucanase. Endo5a breaks down cellulose by cleaving the 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 performed codon optimization for the expression in Escherichia coli, and the sequence was synthesized by IDT for us.

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]