Library selection experiment
The purified plasmid library was electroporated into ΔybeX::kan .
The cells were recovered in LB medium for 1 hour at 37°C 850 rpm shaking
thermostat (Eppendorf Thermomixer compact). The recovery culture was
diluted in LB medium supplemented with 50 µg/mL Zeocin and grown for 12
hours at 37°C 200 rpm shaking (Infors HT Minitron shaker). After 12
hours, the cell cultures were diluted 100x in fresh LB containing 50
µg/mL Zeocin and grown for an additional 12 hours at 37°C. The previous
step was repeated, and the plasmids were purified at every stage (12h,
24h, and 36h). The purified plasmid pools were cleaved via FastDigest
SfiI (Thermo Scientific) restriction enzyme at 50°C. Excision of the ORF
inserts of the plasmid pools revealed in agarose electrophoresis several
bands, of which the most prominent was slightly over 500 kb
(Fig. S8c ). The enriched plasmid pools were transformed in DH5α
Inoue chemical competent cells (Green and Sambrook, 2020) and plated on
LB agar plates containing 25 µg/mL Zeocin (Invivogen). Colony PCR was
performed using ISM1 (GGC TTG GCC CTG AGG GCC) and ISM2 (GTG GCG GCC GCA
TAG GCC) primers following manufacturer protocol for Hot
FirePol® blend master mix ready to load (Solis
BioDyne, #04-25-00115). The PCR cycling parameters were 95°C 12 min,
(95°C 20 sec, 56°C 60 sec, 72°C 20 sec/kb) × 28 cycles, 72°C 10 min, and
15°C infinite hold. PCR products were sequenced via Sanger sequencing
(University of Tartu).