Assembly of genetic parts for CRISPR mediated interference against antimicrobial resistance

This thesis presents parts of a collaborative project designed by a team of the University of Padua that is going to participate to the iGEM competition on synthetic biology. The project aims to exploit the CRISPR interference system to contrast antimicrobial resistance (AMR). The idea we aim to develop consists in delivering a CRISPR interference system, targeting antibiotic resistance genes, by engineered bacteriophages. These phages will be modified to specifically infect one of the following hosts: Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. For a hypothetical application, the platform would be used in a synergic approach with antibiotic therapy to treat infections caused by antibiotic resistant bacteria. In particular, the thesis deals with the construction of a dCas9 and sgRNAs expression systems able to function in the above mentioned bacteria. Specifically, plasmids described in the scientific literature were engineered, mainly through PCR mutagenesis and the BioBrick RFC [10] standard assembly. The use of this standard required the mutagenesis of the dCas9 used in the project. The creation of these standardized parts, promoted by iGEM itself, aims to simplify updates of target genes and bacterial hosts for future implementations, raising a possible solution to the global issue of AMR.