Towards the construction of a chitin-binding domain BioBrick®: PCR Amplification of the Pseudomonas aeruginosa chiC chitin-binding domain

Abstract

Historically, the chitin-binding domain (CBD) of the PA01 strain of Pseudomonas aeruginosa, Chitinase C (ChiC), has been employed as a protein tag in purification processes. However, there remains a need for CBD tags with new properties, such as increased temperature stability or binding affinity. The CBD of PAO1 ChiC is a novel CBD showing promise for purification assays as it could have the aforementioned properties. This study aimed to design a gene block of the PAO1 ChiC CBD and insert it into an expression vector to confirm CBD functionality separate from its native protein and its potential as a tag. This gene block was also designed for cloning into a BioBrick® vector, a standardised vector for interchangeable parts. Insertion into a BioBrick® would allow a standardised protocol for tagging proteins with a CBD, through different combinations of BioBrick® parts. Furthermore, because the cysteine residues within the CBDs of other organisms have been critical to proper domain folding, the functional relevance of the sole ChiC CBD cysteine in PAO1 was also explored. To do this, a second gene block was designed with the substitution of alanine for cysteine. This study describes the experimental design for cloning a CBD-encoded gene block into a pET-28a(+) plasmid, evaluating its functional activity using a chitin-binding assay and then cloning the gene block upon functional activity verification into a BioBrick®. In this study, we were able to design these gene constructs and experimentally showed two primers that could amplify the gene construct. These findings have promising implications for the future as the gene constructs could be inserted into a vector. There are then implications for researchers to use this CBD in the assembly of unique protein sequences which could have applications in producing chitin-based materials for use in biomedicine and agriculture.