Part:BBa_K3686008

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]

Description

Chitin is a natural polymer, present in fungal cell wall, cuticles and exoskeletons of invertebrates. That can also be produced by insects as a protective layer covering their pre-gut (part of digestive system) to shield the epithelial cells. Where the damage of this lining can slow down the nymphal development and lethal for some individuals. Chitinase is a catalase produced by many species, which catalyze the hydrolysis of glycosidic bonds in chitin and degrade into their monomers: N-acetyl-D-glucosamine (NAG). Thus, can potentially be used as a biological insecticide in agriculture. Whereas in our project, we intend to synthesis chitinase to increase to toxicity of Bt toxins. Isaria fumosorosea is an insect pathogenic fungus which secrete chitinase to break through hosts’ cuticle, and may have a potential to be used for pest-control. The gene ifchit1 (~1.9kbp) isolated from this organism, coded for a chitinase (ifcht) with molecular mass ~46kDa. Since this fungus grows at a lower temperature (20℃), this chitinase may have a lower optimum temperature than ones with isolated from bacteria.

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.

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.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

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