TPA Screen 3
Gene blocks were synthesized by Azenta (Chelmsford, MA) and prepared as a pooled master mix (10 ng/L for each fragment). Fragments were assembled into the pBAD24 vector using HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA) with 1-hour, 50 C incubation and 0.2 pmol total fragments. After transformation, half of the recovery culture was plated on LB agar with carbenicillin to evaluate transformation efficiency, and the remaining half used to inoculate an overnight culture (LB with carbenicillin) for purification of the combinatorial plasmid library. The assembled combinatorial library was transformed into emrE E. coli via electroporation, prepared for resistance assays as described above, and plated as 10-fold serial dilutions on LB with 120 M cetrimonium bromide, 20 mM TPA+, or neither, without additional antibiotics. Plates with >1000 isolated colonies were resuspended, and the population plasmid DNA was isolated. This number of colonies corresponds to a >95% chance that there is a clone present for each of the 128 variants in the input library34. Illumina adapters were added via PCR using the following primers: Forward Illumina Adapter: 5-ACACTCTTTCCCTACACGACGCTCTTCCGATCTXXXX-3Reverse Illumina Adapter: 5-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTXXXX-3Next generation (Illumina) sequencing was performed by Azenta (Amplicon-EZ). For each experiment, >135,000 quality reads were obtained, corresponding to >1000-fold coverage of each variant in the unselected population, which is sufficient for a selection of this kind.