Isopropyl β d 1 thiogalactopyranoside (iptg)

M. xanthus and E. coli cultures were grown overnight, pelleted and resuspended in CF at OD₆₀₀ ~5. 100 µl of M. xanthus cell suspension (WT and mutants) were mixed with 100 µl of E. coli cell suspension in a 24-well plate. In each well, 2 ml of CF medium supplemented with CPRG (Sigma Aldrich, 20 µg/ml) and IPTG (Euromedex, 50 µM) were added to induce lacZ expression. The plates were then incubated at 32°C with shaking and pictures were taken after 24 and 48 hr of incubation. To test the contact-dependance, a two-chamber assay was carried out in a Corning 24 well-plates containing a 0.4 µm pore polycarbonate membrane insert (Corning Transwell 3413). This membrane is permeable to small metabolites and proteins and impermeable to cells. E. coli cells were inoculated into the top chamber and M. xanthus cells into the bottom chamber.

The coding sequences of the selected nanobodies in the vector pHEN1 were subcloned into a bacterial expression vector pET26b (Novagen) encoding an N-terminal pelB signal sequence plus a C-terminal His Tag using NcoI and NotI restriction sites. Transformed E. coli BL21 (DE3)/pLysS cells expressed nanobodies in the periplasm after overnight induction in 2YT broth with 0.5 mM IPTG (Euromedex, #EU0008-B) at 16 °C. Bacteria were pelleted by centrifugation, resuspended in PBS buffer containing 300 mM NaCl, 1 mg/ml polymyxin B sulfate (Sigma-Aldrich, # 5291-1GM) and Complete™, EDTA-free Protease Inhibitor Cocktail (Roche, #11873580001) and incubated at 4 °C for 1 h with gentle shaking. Periplasmic extracts were obtained by centrifugation (7400 × g, 10 min, 4 °C). Purified nanobodies were isolated on Co + + affinity columns from periplasmic extracts, according to the manufacturer’s instructions, followed by size exclusion chromatography (SEC) with a Superdex 75 column (Cytiva).

TgCPSF4 (277–445) was codon optimized for E. coli, synthetized and cloned by Genscript within a modified pET30-a (+) vector (Addgene) in order to possess an N-Terminal, TEV cleavable, 8*His Tag. Expression of the recombinant protein was performed in BL21(DE3) chemically competent cells. Briefly, on day 1, 50 μl of BL-21(DE3) cells were incubated with 1 ug of plasmid for 10 min at 4°C, transformed by heat shock at 42°C for 45 s and further incubated 10 min on ice. Following transformation, 600 µl of Luria Broth (LB - Formedium) were added and a 1 hr, 37°C pre-culture was undertaken before platting 150 µl of pre-culture on LB/Chloramphenicol (Chlo – Sigma Aldrich)/Kanamycin (Kan - Sigma Aldrich) agar plates which were further incubated 12 hr. On day 2, a single colony was harvested to inoculate 50 ml of LB/Chlo/Kan for 12 hr at 37°C. On day 3, the 50 ml saturated pre-culture then inoculated 3*1L of Terrific Broth/Chlo/Kan (TB - Formedium) expression culture (using a 2 ml inoculum) which was incubated at 37°C. Upon reaching an OD600 of 0.5–0.8, cultures were ice cooled to 20°C for 10 min then induced with 500 μM of IPTG (Euromedex) for 12 hr after which cultures were centrifuged and stored as dry pellets at −80°C.