To investigate whether the glyco-colicins were capable of inducing aggregation of E. coli, the BW25113 WT strain was cultured in static, anaerobic conditions using glycerol as a primary carbon source in place of glucose to promote phase on transitions resulting in fimH expression. Incubation of the bacteria with increasing concentrations of both (Gly-Ser)1 and (Gly-Ser)3 mannose glyco-colicins was then performed with concentration dependant bacterial aggregation observed by confocal microscopy for both linker lengths (Figure ), with no aggregation observed when ΔfimH and ΔbtuB knockout strains were used (Figure ). The extent of cell aggregation in each sample was quantified by measuring aggregate particle dimensions, surface area and two cross sectional measurements (termed 'width' and height') across three experimental replicates in WT, ΔfimH and ΔbtuB cells in the presence and absence of 1 mM of both mannose glyco-colicins (Figure ). Increasing levels of aggregation was observed in cells incubated with increasing concentrations of both conjugate in contrast to ΔfimH and ΔbtuB where no significant aggregation was observed irrespective of conjugate concentration (Figure ). Quantitative assessment of aggregation further re-enforced this observation demonstrating unequivocally the efficacy of the two conjugates trigger aggregation in cells presenting FimH and BtuB at their surface with statistically significant positive shifts, quantified via application of a Mann Whitney U t-test to subject populations in the aggregate surface area distributions (Figure ), observed for WT cells in the presence of 1 mM of both conjugates ((Gly-Ser significant change in ΔfimH and ΔbtuB aggregate surface area distributions was observed upon incubation with 1 mM glycocolicin.