Wet Lab
Experimental Workflow and Goals
The wet lab team’s fundamental purpose for this project is to test and validate generated RBD (receptor-binding domain) sequences generated by the dry lab team in vitro. While computational models can predict RBD–ligand interactions, in vitro experiments are required to confirm functional activity and ensure that predicted efficacy translates into measurable biological and therapeutic outcomes.
To assess the in vitro functionality and efficacy of candidate RBDs, we leverage the fact that the sequences coding for them are, in general, highly modular for phages, allowing them to be interchanged with each other without disrupting the entire viral particle. Thus, we endeavour to swap the wild-type (WT) RBD sequence within a given phage with our generated one, which enables us to test the latter’s ability to bind to bacterial targets with specific receptors of interest. This will be done through a combination of homologous recombination and CRISPR-Cas counterselection.
To achieve this, two plasmids are co-transformed into a bacterium containing a prophage (an E. coli K12 lysogen). The first plasmid contains the generated RBD with homology arms flanking the upstream and downstream regions of the prophage RBD – this will undergo recombination to swap the generated RBD into the prophage, replacing the WT counterpart. This process has very low efficiency, which necessitates the use of a second plasmid containing Cas machinery and an sgRNA that targets the WT RBD. The sgRNA complexes with the Cas enzymes to cut prophages containing the WT RBD sequence selectively, which has a twofold benefit of counterselecting against non-recombined prophages and creating double-stranded breaks that facilitate the RBD recombination/swapping process. Following this, the swapped phages are rebooted, and efficacy is then evaluated by phage spotting assays, where successfully engineered phages are expected to bind glycans or proteins that WT variants cannot recognize.
The workflow described in the following pages is divided into two main sections: plasmid preparation and phage preparation. The plasmid preparation stage describes the construction and assembly of the sgRNA- and RBD-containing plasmids. Once sequencing confirms that both have been assembled correctly, they are then co-transformed into K12 lysogen cells, which transition over to the phage preparation stage. Swapping and phage rebooting takes place within K12, where phages are extracted and their RBD specificity is assessed through phage spotting.
