Coding

Part:BBa_K3394000:Design

Designed by: Talia Shaler   Group: iGEM20_BGU-Israel   (2020-10-05)
Revision as of 09:15, 5 October 2020 by Talia sh (Talk | contribs)


Coding Sequence of Endo5a cellulase (for E. coli)


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]


Design Notes

We verified that the sequence is optimized for use in Escherichia coli. 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).


Source

Paenibacillus sp. ICGEB2008. NCBI Genbank number for Endo5a is HQ657203.1 (DNA), AEB00655.1 (amino acid).

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. The microorganism that exhibited the most CMCase activity on the agar plate containing CMC was characterized as a Paenibacillus sp. (Paenibacillus ICGEB2008) by phylogenetic analysis of its 16S rRNA gene. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The Endo5A enzyme cloned from Paenibacillus ICGEB2008 contained a catalytic domain associated with glycosyl hydrolase family 5. The enzymes classified into this family are typically present in cellulolytic bacteria and fungi.

References