On the dual effect of glucose during production of pBAD/AraC-based minicircles
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Author(s)
Simcikova, Michaela
Monteiro, Gabriel A.
Prather, Kristala L. Jones
Prazeres, Duarte M. F.
Date Issued
March 2014
Journal
Vaccine
Publisher
Elsevier
Citation
Simcikova, Michaela, Kristala L.J. Prather, Duarte M.F. Prazeres, and Gabriel A. Monteiro. “On the Dual Effect of Glucose During Production of pBAD/AraC-Based Minicircles.” Vaccine 32, no. 24 (May 2014): 2843–2846.
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Author's final manuscript
Abstract
Minicircles are promising vectors for DNA vaccination, gene or cell therapies due to their increased transfection efficacy and transgene expression. The in vivo production of these novel vectors involves the arabinose inducible excision of a parental molecule into a minicircircle and a miniplasmid bacterial backbone. Tight control of recombination is crucial to maximize minicircle yields and purity. In this work, a minicircle production system was constructed that relies on the enzymatic activity of ParA resolvase, a recombinase that is expressed under the transcription control of the arabinose inducible expression system pBAD/AraC, and on Escherichia coli BWAA, a strain improved for arabinose uptake. Undesired recombination already after 4 h of incubation in Luria-Bertani broth at 37 °C was observed due to the leaky expression from pBAD/AraC. While addition of glucose to the growth media repressed this leaky expression, it triggered a pH drop to 4.5 during exponential phase in shake flasks, which suppressed growth and plasmid production. The quantitative PCR analysis confirmed only few copies of high-copy number plasmid inside of the E. coli cells. To ensure the stability of minicircle-producing system, seed cultures should be grown at 30 °C with glucose overnight whereas cells for minicircle production should be grown in shake flasks at 37 °C without glucose up to early stationary phase when the recombination is induced by addition of arabinose.
MIT Department
MIT-Portugal Program
Massachusetts Institute of Technology. Department of Chemical Engineering
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Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/j.vaccine.2014.02.035
