Fig. 1.

Structure of encoded fusion proteins and plasmid construction scheme used in this study. Plasmid sizes are depicted below the plasmid names. Structure of the encoded fusion proteins are shown schematically, with CtxB signal peptide in yellow, 6xHis epitope in black, passenger domains in light red, epitope and protease cleavage site in violet, EhaA autotransporter in blue/green, and AIDA-I autotransporter in orange/pink. FXa protease recognition site is indicated with an asterisk (*). Two asterisks (**) indicate the amino acid sequence encoded by the multiple cloning site. 1=Commercial synthesis in the pJExpress backbone by DNA2.0 (Menlo Park, CA, USA), 2=amplification of pMATE-MT004 backbone using SI020 and PQ019 primers, 3=amplification of mCherry from pEF1a-mCherry-C1 using SI021 and PQ024 primers, 4=amplification of pMATE-SI015 backbone using PT-MCE-7f and PT-MCE-8r primers, 5=amplification of AIDA-I β-barrel and linker region from pES01 using PT-MCE-5f and PT-MCE6r primers, 6=amplification of pMATE-MT004 backbone using PT-MCE-3r and PT-MCE-4f primers, 7=amplification of estA from pES01 using PT-MCE1f and PT-MCE2r primers. The respective PCR fragments were combined using In-Fusion cloning technique (Clontech Laboratories Inc, Mountain View, CA, USA). pES01 has been described previously by Schultheiss et al. (31)