Vibrio Cholerae CRISPR-cas PAM requirements
Vibrio Cholerae CRISPR-cas PAM requirements
1.0 µL from each pellet was resuspended in 20µL dH2O and incubated at 95˚C for 10 minutes.    PAM libraries were transformed into E. coli S17.  E. coli S17 + PAM libraries were grown in LB with 30 µg/mL Chloramphenicol to OD600 1.0, and then pooled equally.  The E. coli  S17 +PAM libraries were introduced into V. cholerae strains 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.  The plates were scraped the next day, and cells were resuspended in LB and pelleted.  From each cell resuspension, 1.0 µL was used as a template in a PCR reaction with universal forward oligonucleotide and reverse index oligonucleotides.  From each cell resuspension, 1.0 µL was used as a template in a PCR reaction with universal forward oligonucleotide and reverse index oligonucleotides.