Fig. 3: Establishment and utilization of ReaL-MGE 1.0 in E. coli BL21. | Nature Communications

Fig. 3: Establishment and utilization of ReaL-MGE 1.0 in E. coli BL21.

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

Fig. 3: Establishment and utilization of ReaL-MGE 1.0 in E. coli BL21.The alternative text for this image may have been generated using AI.

a Diagram of pBBR1-PRha-Redγβα-xseB-PBAD-Cas9-xseAi-Km, with details of the xseA inactivation cassette shown in the inset. The xseA gene in the genome is illustrated, including the target sequence (TS, blue) and inactive PAM version on the inactivation cassette (synonymous PAM mutation, SPM, red). b Five protocol variations (i – v) are illustrated based on twice electroporations with different combinations of gRNA-expressing PCR fragments and dsDNA substrates, arabinose induction was applied in two recovery periods. c Single-mutagenesis efficiencies (500 bp genomic region replaced by 230 bp dsDNA substrate) using different protocols as indicated, dReaMGE plus (dReaMGE with ExoVII exonuclease modified). d Triple mutagenesis efficiencies of E. coli using (i–v) and different dsDNA multiplex applications (dsDNA recombineering, dReaMGE, dReaMGE plus and CREATE). 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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