Constructing CRISPR Plasmids for Gene Regulation

We used a pKH6 vector from Dr. Stephen Lory of Harvard Medical School^{22 (link)} to construct pKH6-CRISPR2 and pKH6-CRISPR1 plasmid. These plasmids were each transfected into PA14 containing pKH-t4rnl1, respectively. Overnight cultures of PA14 WT with pKH13-t4rnl1 (or pKH13-t4K99N) and pKH6-CRISPR2 (or pKH6-CRISPR1) plasmid were grown in LB broth with antibiotic and diluted the overnight culture to OD₆₀₀ = 0.01. When OD₆₀₀ = 0.5, IPTG was added for 1 h incubation and then added the l-arabinose to 20% for different time points. The cells were centrifuged at 12,000 rpm/min for RNA isolation with DNase treatment. One microgram of total RNA was converted to complementary DNA (cDNA) was synthesized with SuperScript III First-Strand Synthesis system (Invitrogen). RT-PCR were performed using GoTaq Green Master Mix (Promega) with specific primers (Supplementary data 3). The PCR products were recovered and cloned into a pMD-19 vector (Takara) for sequencing.