Tracking Synthetic Adhesins on Bacterial Surfaces with Immunofluorescence Microscopy

Programmable bacterial adhesion enables applications in biocatalysis, living materials, and targeted drug delivery. This functionality can be achieved through the expression of synthetic adhesins in bacterial cells. These molecules are hybrid proteins composed of four key elements: (i) a signal peptide for secretion; (ii) the beta barrel domain of an autotransporter for surface display; (iii) a nanobody module providing specific recognition and targeting capabilities; and (iv) an epitope tag for expression tracking and localization. This chapter describes a detailed protocol for visualizing these synthetic adhesins on Escherichia coli and Pseudomonas putida using indirect immunofluorescence combined with Super-Resolution Microscopy (SRM) techniques, such as STimulated Emission Depletion (STED). The workflow includes plasmid introduction into the bacterial cells, induction, immunostaining, and image acquisition to spatially track the adhesins on the cell surface.