Fig. 1: Addition of Cas9 expression and gRNA-expressing PCR fragments to dReaMGE in E. coli BL21. | Nature Communications

Fig. 1: Addition of Cas9 expression and gRNA-expressing PCR fragments to dReaMGE in E. coli BL21.

From: ReaL-MGE is a tool for enhanced multiplex genome engineering and application to malonyl-CoA anabolism

Fig. 1: Addition of Cas9 expression and gRNA-expressing PCR fragments to dReaMGE in E. coli BL21.The alternative text for this image may have been generated using AI.

a The plasmid, pBBR1-PRha-Redγβα-PBAD-Cas9-Km, was used for rhamnose induction of the Red operon (Redγ, β, α) and arabinose induction of Cas9. b The steps in the experiment are illustrated. Step 1: ① rhamnose induction; ② co-electroporation of three asymmetrically 5’ phosphorothioate dsDNA HR substrates (illustrated in the box with S denoting phosphorothioate, P denoting phosphorylation), each with 100 bp homology arms (red, purple or green denoting the different chosen genomic target sites); ③ addition of media for recovery in tube. Step 2: ④ co-electroporation of three gRNA-expressing PCR fragments protected by symmetrical 5’ phosphorothioate, each driven by the J23119 promoter; ⑤ addition of media for recovery in tube before plating. c Optimum titration of gRNA-expressing PCR fragments total input in Step 2 (P = 0.0037). d Examination of varied Cas9 expression by arabinose addition at the indicated points. Using 200 ng gRNA input, the addition of arabinose at ① delivered no improvement over dReaMGE. Arabinose addition in both recovery I ③ and II ⑤ delivered the most benefit for the triple mutation (2.6-fold compared to dReaMGE). Values are means of the biological replicates, and the error bars indicate the standard deviations of all (n = 4) biological replicates. P values were obtained using the two-tailed Student’s t test: ****P < 0.0001.

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