Exploration of protein design-scapes by microfluidic DNA synthesis elucidates domain coupling in the bacterial histidine kinase CpxA

Three plasmid libraries (Tube, Full, Subset) are transformed into chemically competent JW3882-1 (cpxA771(del)kan) obtained from the Coli Genetic Stock Center Molecular (Cellular & Developmental Biology, Yale University). Libraries are plated to onto four 10cm petri dishes with LB (with chloramphenicol and kanamycin) per library and scraped after 18 hours of incubation at 37C. Cells are washed with LB media and freezer stock aliquots are made for subsequent use. To perform screening experiments, single freezer stocks are recovered by shaking in LB at 37C for 4 hours, followed by a single wash in M9 minimal media. Cells are diluted in M9 media to OD600 0.1 and 1 million cells are sorted by Fluorescent activated cell sorting (MoFlo Astrios) using a 100 micron nozzle.