Parts Overview

Our project aims to create a chimeric chitinase by combining substrate-specific domains of naturally occurring chitinases to degrade the chitin of the fungal cell wall. We designed two chimeric bacterial chitinases from three wildtype bacterial chitinase domains, namely chitinases from Serratia marcescens, Amycolatopsis orientalis, and PseudoalteromonasDL-6, and two plant chimeric chitinases using domains derived from Triticum aestivum and Hordeum vulgare wildtype chitinases. We obtained the protein sequences of the respective wildtype chitinases and are reverse-translated using the Expasy tool. The nucleotide sequences are then codon-optimized to express in E.coli. The sequence is made RFC-10 compatible before getting synthesized from IDT, and also restriction sites for BamHI and HindIII are added to clone it in the pET28a vector. In the case of chimeric sequences, either the entire sequence is linked using glycine-serine linker or adding extra Chitin binding domain to catalytic domains of chitinases not having a Chitin binding domain. The second bacterial combination containing chimera of Amycolatopsis orientalis and Serratia marcescens is designed to modify our project. The sequences are optimized using the BOOST Juggler tool for efficient protein production under IPTG induction.