In conclusion, we demonstrate that OPAL bioconjugation can be successfully used to append functional handles such as biotin and fluorescein to colicin proteins. Furthermore, OPAL ligation does not preclude the binding of colicin E9 to its target BtuB receptor on the surface of E. coli, evidenced by fluorescent labelling of living bacterial surfaces characterised using fluorescence confocal microscopy and 3D-SIM. Finally, this work establishes that mannose presenting glyco-colicins can induce aggregation in an adherent E. coli strain, which was validated using fluorescence confocal microscopy and quantified statistical analyses of aggregate sizes. We hypothesise aggregation is a result of simultaneous binding of conjugates to the BtuB outer membrane receptor on one E. coli bacterium and a FimH adhesin on an adjacent bacterium, highlighting the potential future applications of such glyco-colicins as anti-adhesion therapies against pathogenic bacteria where adhesion to host cell epithelial surfaces plays an important role in colonisation