Generation and Characterization of OmpX Variants

Variants of OmpX from E. coli containing loop inserts were generated by blunt-end ligation of PCR products. A pET3b plasmid containing ompX without the signal peptide, a T7 promotor and ampicillin resistance (Pautsch et al., 1999 (link)) or an identical plasmid containing the complete OmpX sequence (with signal peptide (Arnold et al., 2007 (link))) was used as a template for the PCR. Primers were designed to amplify the plasmid with overhangs containing the intended insert. Primers are listed in Supplementary Material S1 (Supplementary Table S1). Ligation products were transformed into calcium-competent E. coli TOP10 cells (Invitrogen) and the DNA sequence of the inserts was confirmed with Sanger sequencing (LightRun, Eurofins).
For in vivo studies of OmpX folding, a variant of E. coli BL21 had to be generated that lacked the wildtype OmpX gene. This was done using P1 transduction as described (Saragliadis et al., 2018 (link)), with E. coli BL21 as the acceptor strain and an OmpX knockout from the Keio collection (Baba et al., 2006 (link)) as the donor strain. This gave rise to the kanamycin-resistant strain E. coli BL21ΔOmpX. Absence of the ompX gene was verified by Western blotting of whole-cell lysates using an OmpX rabbit antiserum (Arnold et al., 2007 (link)) and E. coli BL21 as a positive control (data not shown).