Fastlink kit

Heterologous expression libraries were prepared as previously described^{23 (link)}. In brief, genomic DNA was extracted from overnight cultures of the source bacterial strain^{24 (link)}. DNA was sheared to 2–8 kb by focused ultrasonication (Covaris E220 with miniTUBE red) and fragments were cloned into PCR-linearized expression vector pZE21 (primer #1–2) by blunt-ended ligation (Epicentre FastLinkTM kit). Before transformation, the ligation products were separated on a 0.5% agarose gel, the region between 5 and 10 kb was excised and DNA was extracted using a gel extraction kit (Qiagen). Ligation products were transformed into E. cloni®10G Elite competent cells (Lucigen) by electroporation. Overnight grown colonies were picked and arrayed in 384-well format into liquid LB medium supplemented with kanamycin using a colony picking robot (Molecular Devices QPix 420). After incubation overnight at 37°C, plates were replicated in duplicate onto LB agar plates supplemented with kanamycin. The first plate served for the initial drug assay described below to identify plates that contain drug-metabolizing gain-of-function hits. The second plate was stored at 4°C for use in the secondary assay to localize drug metabolizing clones within active plates as described below. Primer #3–4 were used for Sanger sequencing of representative library clones and identified drug-metabolizing clones.