Phage delivery of CRISPR interference system to aid AMR bacteria sensitization

My thesis project aims to better understand, characterize and improve the use of phage particles as platform for the delivery of the CRISPRi system to re-sensitize AMR E. coli. By leveraging the methods and technologies of synthetic biology, we aim to create innovative solutions that can reduce the spread of antibiotic-resistant bacteria and improve the effectiveness of current antibiotics since the development process required for new antibiotics takes years, costs millions, and can’t keep up with the AMR emergency. Our focus is to render the recombinant phage M13 able to be used as a platform for the delivery of the dCas9 and sgRNA targeting AMR genes in Escherichia coli. To do this, we need firstly to understand and characterize the ability of the phage to be used as a platform by delivering a reporter gene that in our case will be an RFP cassette. Next, we will be testing the system through the delivery of a more complex circuit containing the CRISPRi system against the RFP. Lastly, we will be employing this toolkit to target two AMR genes, the ndm-1 and the mcr-1, respectively for the resistance to Meropenem and Colistin, present in two E.coli laboratory strains.