Figure 1
From: Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline
High-throughput expression and purification of proteins assisted by the OT-2. (a) Several key steps in the high-throughput expression and purification workflow that were automated using the OT-2: transformation of chemically competent E. coli, inoculation from 96-well starter cultures to four 24-well plates for expression, and Ni-charged magnetic bead purification of the recombinant proteins. The purification step involved binding the His-tagged target protein to the magnetic beads, washing away cell debris and non-target proteins, then cleaving the target protein off the magnetic beads with proteolytic cleavage. (b) The workflow began with transformation of a plasmid library into chemically competent E. coli cells, which were then grown to saturation. (c) The 96-well plate starter cultures were used to inoculate four 24-well plates containing 2 mL autoinduction media. The growth of the expression cultures occurred in a standard (19 mm orbit, 300 rpm) shaker-incubator until saturation. (d) The cells were then harvested via centrifugation. (e) The cells were resuspended in a chemical lysis solution and transferred via the OT-2 back to a 96-well plate for purification. The Ni-charged magnetic bead purification protocol, the most tedious step to perform without liquid handling assistance, employed the OT-2 magnetic module and performed washes to remove non-target proteins and add the protease for cleavage. A protease cleavage liberated the target proteins from the beads and the OT-2 performs a final transfer of the purified protein supernatant to a fresh plate for testing. Portions of this figure were created with BioRender.com.
