Fig 2.

CRISPRi knockdown of secondary metabolite production in P. luteoviolacea. (A) Schematic representation of modular CRISPRi parts adapted to include dCas9-bla and Ptac sgRNA parts, pMMK601, and pMMK602, respectively. Part plasmids are combined, and a BsmBI Golden Gate Assembly was performed. (B) Schematic representation of the violacein gene cluster vioABCD in P. luteoviolacea and the violacein molecular structure. The CRISPRi system was assembled with an sgRNA targeting the vioA gene (pMMK603) and employed to knock down violacein production in P. luteoviolacea. (C) P. luteoviolacea with gfp (pMMK815) or vioA (pMMK816) sgRNA plasmids grown on marine agar plates. (D) Quantification of violacein production (measured at 580 nm) between P. luteoviolacea containing gfp or vioA sgRNA plasmids. Asterisks indicate significant differences (*P = 0.02, Dunn’s multiple comparisons test). Bars represent the mean of eight total replicates and error bars indicate standard deviations.