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. 2019 Feb 22;13(1):210–221. doi: 10.1111/1751-7915.13382

Figure 3.

Figure 3

The CRISPRi‐mediated repression of a heterologous gene in Pseudomonas putida KT2440. (A) Schematic representation of CRISPRi targeting the green fluorescent protein (GFP) of pST‐GFP plasmid. The pST‐GFP plasmid expresses GFP under the control of IPTG‐inducible Ptrc promoter. A 20 bp sgRNA spacer sequence targeting the gfp gene of pST‐GFP was designed to bind the non‐template DNA strand 33 bp away from the ATG translational start codon of the gfp gene. (B, C) CRISPRi‐mediated repression of plasmid‐borne gfp gene in P. putida KT2440. The pST‐GFP and pSECRi(GFP) plasmids were co‐transformed into P. putida KT2440, and the transformants were grown on LB medium containing 1 mM l‐rhamnose in the presence (0.1 mM) or absence of IPTG. Cell growth and GFP fluorescence were monitored simultaneously using an Infinite 200 PRO reader for 23 h at 30°C. Each graph represents the mean value of the corresponding optical density at 600 nm (OD 600) or green fluorescence ± standard deviation of duplicate measurements from at least three independent experiments.