Difference between revisions of "Part:BBa K3394000:Design"

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===Design Notes===
 
===Design Notes===
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We verified that the sequence is optimized for use in Escherichia coli, and that the active protein could be expressed on the bacteria's surface.
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We perform codon optimization for the expression in Escherichia coli.
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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).
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===Source===
 
===Source===
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===References===
 
===References===
 
Adlakha, N., Rajagopal, R., Kumar, S., Reddy, V. S., & Yazdani, S. S. (2011). Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium. Applied and environmental microbiology, 77(14), 4859-4866.
 
Adlakha, N., Rajagopal, R., Kumar, S., Reddy, V. S., & Yazdani, S. S. (2011). Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium. Applied and environmental microbiology, 77(14), 4859-4866.
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Gupta, S., Adlakha, N., & Yazdani, S. S. (2013). Efficient extracellular secretion of an endoglucanase and a β-glucosidase in E. coli. Protein expression and purification, 88(1), 20-25.

Latest revision as of 11:28, 22 October 2020


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 perform codon optimization for the expression in Escherichia coli.

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

Adlakha, N., Rajagopal, R., Kumar, S., Reddy, V. S., & Yazdani, S. S. (2011). Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium. Applied and environmental microbiology, 77(14), 4859-4866.

Gupta, S., Adlakha, N., & Yazdani, S. S. (2013). Efficient extracellular secretion of an endoglucanase and a β-glucosidase in E. coli. Protein expression and purification, 88(1), 20-25.