Figure 1
From: Recombineering using RecET in Corynebacterium glutamicum ATCC14067 via a self-excisable cassette
Optimization of linear dsDNA recombineering parameters. (a) Effect of the homology length of linear dsDNA on recombination efficiency. 100, 200, 300, 400, 500, 800, 1000, 1200, 1500, 2000 bp left and right homology arms of the crtB gene combining with the Kan cassette were used for the investigation. 0.5 μg dsDNA cassettes were used for electroporation. The recovery time was 5 h. (b) Effect of the quantity of linear dsDNA on recombination efficiency. 0.1–4 µg CrtB/800-Kan cassettes were used for electroporation. The recovery time was 4 h. (c) Effect of the recovery time on recombination efficiency. The recovery time was 0 h to 5 h. 0.5 μg CrtB/800-Kan cassettes were used for electroporation. The total number of colony of per OD600 also is shown. (d) Effect of the induction time on recombination efficiency. The RecET recombinases were induced for 2–8 h. 0.5 μg CrtB/800-Kan cassettes were used for electroporation and the recovery time was 4 h. OD600 of the harvested culture is also shown. (e) Effect of the phosphothiolated linear dsDNA on recombination efficiency. 0.5 μg CrtB/800-Kan cassettes were used for electroporation. The recovery time was 4 h. All of the Kmr cfu is the number of kanamycin resistance colony per mL. Datas are the means of at least three experiments with standard deviations by error bars.
