0.2 m membrane filters

Overnight cultures (100 ml culture incubated at 37 °C with shaking) from the positive clones, previously supplemented with auto-induction solution and chloramphenicol, were centrifuged at 3500 rpm for 10 min. Afterwards, the cell pellets were re-suspended in 20 ml of 10 mM Tris–HCl pH 7. The extracts were sonicated on ice at 20% maximal amplitude for 370 s, with 10 s intervals without sonication (Branson 150D Ultrasonic Cell Disruptor with 3 mm diameter sonotrode). The extracts were finally filter-sterilized with 0.2 µm membrane filters (Corning) [76 (link)]. Protein concentrations of the extracts were determined by the Pierce™ bicinchoninic acid BCA protein assay kit (ThermoFischer).

Whole-cell extracts were prepared from the clones namely 102-5A and 88-1G, using a method similar to that reported in the literature [47 (link)]. Overnight cultures from the E. coli bearing the eDNA from the positive clones -previously supplemented with auto-induction solution and chloramphenicol- were centrifuged at 3,500 rpm for 10 min. Afterwards, the cell pellets were resuspended in 0.01 M Tris–HCl pH 7. The extracts were then sonicated on ice at 20% maximal amplitude for 370 s (with 10 s intervals without sonication). Finally, the extracts were sterile-filtered with 0.2 µm membrane filters (Corning) [47 (link)]. Two negative controls were used: (1) cells incubated with only the media, and (2) cells incubated with buffer. The buffer concentration, in the latter, was the same concentration used in the highest extract concentration 50% v/v (Fig. 2).