Fig. 1

Preparation of E. coli transformants that displayed LPP′-OmpA′-SUMO-Δ59 Sortase A-LPETG-PEN-HuscFv34-6× His on the surface. (a) Diagram of a DNA construct coding for Lpp′-OmpA′-SUMO-Δ59 Sortase A-LPETG-PEN-HuscFv34. (b) Amplicons of Lpp′-OmpA′ (456 bp), SUMO (288 bp), Δ59 Sortase A (447 bp) and LPETG-pen-huscfv34 (840 bp). (c) Amplicons of Lpp′-OmpA′-Δ59 Sortase A-LPETG-pen-huscfv34 contig (∼2000 bp, arrowhead) in the DH5α competent E. coli clones 1–15 that had been transformed with Lpp′-OmpA′-Δ59 Sortase A-LPETG-pen-huscfv34-pET28b⁺ vector (lanes 1–15, respectively); lanes M of (b) and (c) are DNA size standard; lane N of (c) is negative DNA template control; lane P of (c) is positive DNA template control; numbers at the left of (b) and (c) are DNA sizes in bp. (d) Flow cytometric analysis of transformed BL21 (DE3) ΔA competent E. coli clones 1–5 that were stained by mouse monoclonal anti-HuscFv34 antibody and goat anti-mouse Ig-Alexa Fluor 488 conjugate for detection of Lpp′-OmpA′-SUMO-Δ59 Sortase A-LPETG-PEN-HuscFv34-6× His fusion protein on the cell surface. Cells were counter-stained by DAPI to indicate nuclei. Q1 and Q2, transformed E. coli cells displaying Lpp′-OmpA′-SUMO-Δ59 Sortase A-LPETG-PEN-HuscFv34-6× His fusion protein; Q3 and Q4, E. coli cells that did not display the fusion protein. Q2 and Q3, intact bacterial cells with nuclei; Q1 and Q4, bacterial cells without nuclei or dead cells