Part:BBa_K3686008:Design

Chitinase from Isaria fumosorosea, complete CDs, codon-optimized.

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 NgoMIV site found at 642
  Illegal NgoMIV site found at 646
  Illegal NgoMIV site found at 730
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Codon optimized for expression in E. coli.

Source

Genome of Isaria fumosorosea, full amino acid sequence submitted under Genbank accession number KR184828

Experiment details

The gene ifchit1 has been codon-optimized for expression in E. coli:

GC contant

and attached with a 6*His-group for Ni-NTA purification. Which then sealed onto the vector pET28a and transformed into E. coli BL21 (DH3) strain for pTac regulated production.

The colony PCR result of transformed BL21s, lanes labeled with numbers responding to different colonies, where the band above 1kbp indicated the presence of ifchit1 gene.

However, we failed to identify the desired protein from SDS-PAGE results, as illustrated in fig 2. We believed that the absence of protease inhibitor led to the high rate of cleavage. Codon-optimization errors may also be a factor, which could be investigated by tagging GFP at N’ terminal.

.

.Fig2: SDS-PAGE image of ifcht, after induced by 1mM IPTG with different conditions; left: cultured at 25℃ for 8 hours, right: at 16℃ for 12 hours. The homogenate and Ni-NTA purification resin was compared, the band ~46kDa was absent in all samples.

The chitinase activity analysis has been performed, which no significant difference among chitinase activity in bacteria lysate, Ni-NTA flowthrough and control (lysate of non-recombined E. coli) could be observed. (detailed on our protocols and methods

.