Abstract
The laboratory-scale production of plasmid DNA (pDNA) is hindered by the limitations of shake flasks, such as mass transfer capacity and lack of pH control. Consequently, better schemes for pDNA production in shake flasks are needed. pDNA production can be improved by increasing the amount of biomass, increasing the pDNA yield on biomass (YpDNA/OD), or both. In this study, we characterized the production of three differently sized plasmids (5.4, 6.0, and 7.8 kbp, respectively) cultured in Lysogenic Broth (LB), Terrific Broth (TB), and EnPresso B Plasmid (EBP) culture medium that releases glucose to the broth enzymatically. Higher cell densities, higher acetate accumulation, and higher pDNA titers, were obtained in cells cultured in TB than in those cultured in LB medium. The enzyme-controlled glucose release system resulted in an important increase in cell densities, while the YpDNA/OD increased up to threefold. The most important increase in pDNA titer was observed for the 7.8-kbp plasmid, which raised from 1.6 to 4.3 mg/L in LB and TB medium, respectively, to 26.6 mg/L using the EBP medium. These results show that controlled substrate delivery is useful to increase the production of large-sized pDNA in shake flasks.
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This work was supported by CONACyT grants 183911 and 248926.
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Supplementary Figure S1
Agarose gel (0.8 % W/V in TBE buffer) electrophoresis images. Lane 1 (ML) 6 μL of Gene Ruler DNA 1-kb Ladder (Thermo Fisher, Waltham, MA, USA). Upper labels indicate the name of the plasmid loaded (each lane corresponds to a sample from duplicate experiments). 50 ng of pDNA (measured spectrophotometrically) were loaded on each of these lanes. The gel was stained with Sybr Green (Sigama Aldrich, MO, USA) using 1.5 μL of Sybr Green per 30 mL of gel. The gel was run at 80 V for 30 min. A Samples from cultures in LB medium, B samples from cultures in TB medium, C Samples from EBP medium. (GIF 186 kb)
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Galindo, J., Barrón, B.L. & Lara, A.R. Improved production of large plasmid DNA by enzyme-controlled glucose release. Ann Microbiol 66, 1337–1342 (2016). https://doi.org/10.1007/s13213-016-1218-2
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DOI: https://doi.org/10.1007/s13213-016-1218-2