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A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

Hassan, S; Keshavarz-Moore, E; Ward, J; (2016) A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy. Biotechnology and Bioengineering , 113 (9) pp. 2064-2071. 10.1002/bit.25971. Green open access

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Abstract

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from 3 different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total % supercoiled monomer was maintained to 85-90%. A two-fold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. This article is protected by copyright. All rights reserved.

Type: Article
Title: A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy
Open access status: An open access version is available from UCL Discovery
DOI: 10.1002/bit.25971
Publisher version: http://dx.doi.org/10.1002/bit.25971
Language: English
Additional information: © 2016 The Authors. Biotechnology and Bioengineering. Published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Keywords: Cell engineering, E. coli, Fermentation, Plasmid DNA, Segregational stability, Supercoiling density
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/1483523
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