Verification of tn-mutant library complexity by Linker PCR

The complexity of the tn-mutant library was confirmed using a method known as Linker PCR, as described by Christiansen et al.¹⁸. Random selection of ten colonies from BHI-A plates containing 5 mg/L erythromycin, were cultured in BHI broth at 37 °C overnight. DNA was extracted from these overnight cultures using the DNeasy Blood & Tissue kit (Qiagen), with the addition of 0.5 mg/mL (250 units/mL) of lysostaphin (Sigma Aldrich). The DNA was then digested with the RsaI restriction enzyme (Thermo Fisher Scientific) at 37 °C for 16 h, followed by purification using the GenJet PCR Purification Kit (Thermo Fisher Scientific).

Oligo 254 and 256 adaptors (Supplementary Table S4) were prepared by resuspending them in an annealing buffer composed of 100 mM Tris-HCl (pH 8), 500 mM NaCl, and 20 mM EDTA, and then diluting each adaptor to a concentration of 100 µM. The adaptor solutions were combined in a 1:1 ratio and denatured at 95 °C for 3 min, followed by an hour-long incubation at room temperature. The annealed adaptors were then ligated to the digested DNA using T4 ligase (Thermo Fisher Scientific) at 22 °C for an hour. Post-ligation, DNA was purified using the GenJet PCR Purification Kit (Thermo Fisher Scientific) and subjected to PCR using Phusion high-fidelity DNA Polymerase (Thermo Fisher Scientific) with specific primers (forward TnL and reverse primer 258) (Supplementary Table S4). The PCR conditions were as follows: an initial denaturation for 3 min, followed by 30 cycles of 45-sec denaturation at 94 °C, 1 min annealing at 53 °C, and 2 min elongation at 72 °C, with a final 10 min elongation at 72 °C. PCR products were then visualized on a 1% agarose gel run at 90 volts for 30 min.