A modular plasmid system for expression of genes in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Antimicrobial resistance in N. gonorrhoeae is of increasing concern and there is no current vaccine. Over the last 80 years, N. gonorrhoeae has become highly resistant to many frontline antibiotics, and as such, there are now limited treatment options. Research and development is critical for the discovery of new diagnostic assays, antimicrobials, and vaccines that combat N. gonorrhoeae. To date, there are a variety of genetic tools available for generating mutations, gene deletions, and gene complementation in N. gonorrhoeae. Plasmids are available for the introduction of new genes of interest and complementation, however replicating plasmids are often unstable, time consuming to manipulate, and suffer from poor transformation efficiencies.

Here, this project aimed to resolve the current issues with N. gonorrhoeae plasmid systems by using a Golden Gate assembly technique to design a modular cloning system specifically to enable custom gene expression in N. gonorrhoeae. Golden Gate cloning uses Type IIS restriction endonucleases to allow the assembly of complex multipart plasmids from reusable DNA parts. These include the gonococcal-specific cryptic plasmid maintenance region from pEG2, the Opa promoter, antibiotic resistance genes encoding kanamycin and erythromycin resistance, fluorescent proteins, and a ColE1 origin of replication for propagation in Escherichia coli. Our system was designed to be more efficient by enabling the rapid generation of very small, customised plasmids capable of replication in both E. coli and N. gonorrhoeae. Currently, there are vast gaps in our knowledge of N. gonorrhoeae biology. This thesis offers a new system that is an efficient tool for custom gene expression in N. gonorrhoeae to improve our understanding of this pathogen. Improving research approaches, such as mutagenesis and complementation, will contribute to drug and vaccine discovery efforts searching for reliable treatments for this devastating pathogen.