Generating CFTR Variant Library

CFTR variants were generated using the SPRINP mutagenesis method (SI Appendix, Table S2) (54 (link)). Briefly, mutagenic primers were designed to be complementary to the template plasmid except for the mutated bases and to be 15 to 45 nucleotides in length. Plasmid containing CFTR cDNA was amplified in separate reactions containing forward or reverse primer. The single-primer products of these reactions were combined and denatured at 95 °C for 5 min and gradually cooled to 37 °C over the next 5 min. The sample was then digested by DpnI for 4 h. Then, 5 µL of sample was added to 50 µL of competent XL2Blue cells for transformation and incubated on ice for 30 min. The bacteria were then heat-shocked at 42 °C for 45 s and allowed to recover on ice for 2 min. Following that, 200 µL of warmed SOC media (Invitrogen) was then added directly to the cells, and the mixture was allowed to shake at 225 RPM in a 37 °C incubator for 30 min. Then, 200 µL of this mixture was spread on LB/ampicillin plates and left to incubate at 37 °C overnight. Random colonies were then picked and expanded in LB/ampicillin. Plasmid DNA was then purified (QIAGEN Plasmid Kit) and sequenced (Genewiz).