Use of colicin E9 to label E. coli with biologically relevant motifs necessitates the conjugation of the colicin to the desired motifs using a site-specific bioconjugation approach. The Nterminal T-domain of colicin E9 has been shown to be a structurally disordered region with a high degree of flexibility and distal to the colicin E9 binding site. We therefore hypothesized that modification at this site would not preclude binding of colicin E9 to its receptor. Conveniently, the N-terminal residue of colicin E9 is a serine, and thus the installation of a bioorthogonal α-oxo aldehyde motif at this site was easily achieved through a mild treatment with NaIO4 (Figure ). This α-oxo aldehyde could subsequently be targeted through OPAL to sitespecifically append a desired chemical motif to the N-terminus of colicin E9 using proline tetrazole as an organocatalyst (Figure ). To demonstrate that OPAL conjugation to the N-terminus of colicin E9 was possible, a sample of α-oxo aldehydefunctionalised colicin E9 was subjected to OPAL with a biotinpresenting OPAL probe. Successful ligation was validated using both SDS-PAGE gel and western blot analysis (Figure ). Due to the size of colicin E9 (61 kDa) the addition of the biotinylated OPAL probe only results in a subtle observable increase in the mass of the colicin E9 band in SDS-PAGE (Figure ), yet western blot analysis unequivocally demonstrated that the biotin had been successfully attached (see Figure ). To prove that conjugation via OPAL does not prevent binding of colicin E9 to the BtuB receptor, a fluorescein-labelled colicin E9 conjugate was also prepared enabling BtuB binding to be visualised via fluorescence microscopy (Figure ). The successful labelling of colicin E9 with fluorescein was also confirmed via SDS-PAGE gel analysis (Figure ), in which a subtle mass increase can be observed after bioconjugation of the fluorescein-labelled OPAL probe, confirmed by fluorescent visualisation (Figure ).