Pooled Bacillus subtilis transconjugants: sample of trasnconjugants generated via mating with a library of ICEBs1 donor strains
A library of B. subtilis strains was generated, wherein each strain each comprised a CRISPRi knockdown of a single essential B. subtilis gene as well as the integrative and conjugative element ICEBs1. The single guide RNA was incorporated into ICEBs1 itself. Strains were induced to mate with an ICEBs1-lacking recipient strain, and transconjugants were selected for via antibiotic marker selection. Genomic DNA from the pooled transconjugants was extracted using Qiagen DNeasy Blood and Tissue kits according to manufacturer instructions. A portion of the genomic locus encoding the 20 nt variable region of the single guide RNA was amplified by PCR (20 cycles) and purified via SureSelect AMPure beads according to manufacturer instructions. Indexed samples were pooled and sequenced with an Illumina MiSeq using a 20 bp read length using a custom sequencing primer.