Vibrio cholerae TnSeq: ShyA(L109K) toxicity
TnSeq was conducted as described before(42,43); Briefly, cultures of strains MA1 and MA73 were mated with donor strain E.coli MFD  pir, which contains the pSC189 suicide plasmid encoding the mariner transposon. Approximately 200,000 colonies/replicate were recovered from plates containing Kan50 to select for transposon insertions and 1mM IPTG to allow the expression of ShyAL109K. Libraries were then resuspended in 30 mL of LB broth, and 1/5 of the culture was used for genomic DNA (gDNA) extraction and the rest was frozen in 30% glycerol at -80C. Samples were prepared for sequencing as follows. The extracted gDNA was sheared by sonication (9 seconds, 30% amplitude), followed by blunting (Blunting Enzyme Mix, NEB), A-tailing, ligation of specific adaptors and PCR amplification of the transposon-DNA junctions using transposon- and adaptor- specific primers. Libraries were sequenced using Illumina MiSeq as described previously (44). To determine gene essentiality, data analysis was performed using the Matlab-based pipeline ARTIST(43). Genetic regions predicted to be conditionally essential/enriched were inspected using the genome browser Artemis(45) and insertion plots were generated using the tidyverse package in R.