PCR Amplified pTevSpCas9 sgRNA locus from oPool targeting pTox in Escherichia coli
A two-plasmid enrichment experiment was used to assay sgRNA activity. For liquid selections, 50 ng of the sgRNA plasmid pool was transformed into 50 L E. coli NEB 5-alpha FIq competent cells harbouring pTox by heat shock. Cells were allowed to recover in 1 mL of non-selective 2xYT media (16 g/L, 10 g/L yeast extract, and 5 g/L NaCl) for 30 minutes at 37C with shaking at 225 rpm. The recovery was then split and 500 L was added to 500 L of inducing 2xYT (0.04% (w/v) L-arabinose and 50 mg/mL chloramphenicol) or to 500 L of repressive 2xYT (0.4% (w/v) D-glucose and 50 mg/mL chloramphenicol) and incubated for 90 min at 37C with shaking at 225 rpm. The two cultures were washed with 1 mL of inducing media (1x M9, 0.8% (w/v) tryptone, 1% v/v glycerol, 1 mM MgSO4, 1mM CaCl2, 0.2% (w/v) thiamine, 10 mg/mL tetracycline, 25 mg/mL chloramphenicol, 0.4 mM IPTG) or repressed media (1x M9, 0.8% (w/v) tryptone, 1% v/v glycerol, 1 mM MgSO4, 1mM CaCl2, 0.2% (w/v) thiamine, 10 mg/mL tetracycline, 25 mg/mL chloramphenicol, 0.2% (w/v) D-glucose) respectively before addition to 50 mL of the same media that was used in the wash in a 250 mL baffled flask. These cells were grown overnight at 37C with shaking at 225 rpm. Plasmids were then isolated using the Monarch Plasmid Miniprep Kit (NEB) according to manufactuerers specifications. The sgRNA locus was then PCR amplified using primers (Supplementary Table S8) containing Ilumina adapter sequence, 4 random nucleotides, 12-mer barcodes to specify the replicate, and plasmid-specific nucleotides at the 3 end. The resulting amplicons were sent for 150 bp paired-end Illumina MiSeq sequencing at the London Regional Genomics Center (London, ON).