Figure 2.

A Papillation Assay for Spacer Acquisition. (A) A constitutive promoter drives transcription through the CRISPR leader–repeat region into lacZ, which is initially inactive due to an in-frame stop codon. Spacer acquisition restores the reading frame, activating LacZ expression. (B) The assay is performed on LB agar containing 0.1% lactose and X-gal. Only colonies acquiring a spacer develop blue Lac⁺ papillae through lactose metabolism and X-gal cleavage. (C) Two Cas1–Cas2 plasmids, pRC1656 (P_araBAD) and pRC2747 (P_araMari), were introduced into RC5311. Arabinose concentration was varied to modulate expression. Papillation peaked at 0.002% l-arabinose for P_araBAD and at 0.2% for P_araMari. Representative colonies are shown. (D) Thirty Lac⁺ papillae were isolated by re-streaking for single colonies. CRISPR arrays were PCR-amplified and analyzed by agarose gel electrophoresis. All isolates displayed +1 repeat expansions. Ten representative examples are shown. Spacer sequences were confirmed by Sanger sequencing; uppercase letters denote spacer sequence; the PAM is in bold with the first two residues in lowercase to indicate that they are of genomic origin and not present in the CRISPR array. (E) Cells were passaged twice at 1:1000 dilution, and CRISPR array expansion was assessed by PCR. Bands were visible only at high P_araBAD induction, demonstrating the greater sensitivity of the papillation assay compared to bulk PCR.