NextSeq 500 paired end sequencing; Transcription Termination and Antitermination of Bacterial CRISPR Arrays
Transcription Termination and Antitermination of Bacterial CRISPR Arrays
1.0 µL from each pellet was resuspended in 20µL dH2O and incubated at 95˚C for 10 minutes.   Protospacer plasmids and an empty pKS958 control were transformed into E. coli S17.  All E. coli S17 + protospacer plasmid strains were grown in LB with 30 µg/mL Chloramphenicol to OD600 1.0, and then pooled equally, with the exception of the cultures harboring protospacer plasmids 37, 38 and empty pKS958, where only 1/10th of the amount of cells were added to the pool.  The E. coli  S17 + protospacer plasmid pool was introduced into V. cholerae strains BJO185, 2094, and  2206 by conjugation, as previously described (Box et al., 2015).  100µL of cells from each conjugation were plated on LB plates containing 75 µg/mL kanamycin and 2.5 µg/mL chloramphenicol. Scraped cells were resuspended in 10 mL M9 minimal medium and pelleted by centrifugation.  From each cell resuspension, 1.0 µL was used as a template in a PCR reaction with universal forward oligonucleotide JW10344 and reverse index oligonucleotides JW10345 for BJO185 template, JW10346 for 2094 template, and JW10347 for 2206 template.  From each cell resuspension, 1.0 µL was used as a template in a PCR reaction with universal forward oligonucleotide JW10344 and reverse index oligonucleotides JW10345 for BJO185 template, JW10346 for 2094 template, and JW10347 for 2206 template.