Construction of inducible expression vector pDNLS-22n containing P. aeruginosa PAO1 ChiC

Authors

  • Novia Chan
  • Sagar Pannu
  • Lena Xiong UBC Microbiology and Immunology
  • David Yeung

Abstract

In response to concerns over traditional pesticides, biocontrol agents have been touted as safer alternatives. This has garnered attention for chitinase as a biopesticide due to its insecticidal activity. Chitinases break down glycosidic linkages in chitin, which is an important constituent of the arthropod exoskeleton. As a result, we sought to characterize the expression of chiC which codes for an extracellular chitinolytic enzyme ChiC. We constructed the pDNLS-22n vector to enable inducible expression of Pseudomonas aeruginosa PAO1 ChiC in E. coli. The chiC gene was PCR amplified from the storage vector pGKMS21 and ligated into pET-28a(+) by restriction cloning using the EcoRI and HindIII sites. Sanger sequencing of chiC from pDNLS-22n showed complete sequence identity to the reference gene. We thus established an inducible expression vector for ChiC with a leader peptide at the N-terminus that encodes both a 6xHis tag for protein purification and a T7 tag for detection by immunological techniques. Construction of a chiC expression vector enables future studies on the structure and function of ChiC and establishes a system for studying the potential implications and challenges associated with creating a viable insecticidal alternative to pesticides.

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Published

2022-08-31