Creating a Fluorescent Pseudomonas aeruginosa Biofilm Reporter

Pseudomonas aeruginosa is a Gram-negative bacterium that forms three dimensional communities, called biofilms, within immunocompromised hosts to sustain its infection cycle. Communication within these structures is essential as they enable a synchronised reaction from all cells within the biofilm in response to an environmental stimulus. Biofilms are of particular interest in research as they enable production of virulence factors and provide a level of protection against widely used antimicrobials, leading researchers to use a combination of in vitro and in vivo methods to research them. In this study, we used a combination of biofilm assays and C. elegans imaging of already existing P. aeruginosa strains to design a red fluorescing P. aeruginosa biofilm reporter construct. We then built our desired tool using Gibson Assembly which was transformed into P. aeruginosa using electroporation. The strain was then tested in vitro and in vivo for functionality. The resulting strain appears to produce a strong red fluorescence in response to C4-HSL based upon initial trials. This red fluorescence was then seen in low levels in C. elegans fluorescence microscopy in the lumen indicating QS/biofilm formation was occurring. Moving forward, further studies are required due to a lack of biological replicates on the in vitro and in vivo work. Once that is completed, this tool could be used in a variety of ways such as an indicator when looking at novel antimicrobials.