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Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR

Paper ID Volume ID Publish Year Pages File Format Full-Text
35372 45088 2011 8 PDF Available
Title
Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
Abstract

DNA-based vaccines and gene therapies have rapidly evolved since the first demonstration of in vivo gene expression by naked DNA injection. When optimizing their production, the major goals in the fermentation process are to maximize the specific plasmid yield, as well as plasmid quality, while minimizing plasmid-mediated metabolic burden. Due to this fact, the development of new and fast monitoring techniques able to easily assess plasmid-mediated metabolic burden and process productivity is in demand. In this work, Escherichia coli DH5 alpha harboring plasmid pVAX1-LacZ was grown in semi-defined media using several pDNA induction strategies. The effect of the induction strategy on cell physiology and overall process productivity was monitored using multi-parameter flow cytometry and real-time qPCR. All induction strategies caused cell filamentation and decreased viability at the end of fermentation. The results also suggest that an amino acid limitation with AMP addition induction strategy resulted in the highest specific yields (20.94 mg/g) and, concomitantly, highest plasmid copy number (1070 per cell). In conclusion, amino acid limitation with AMP addition seems to be a suitable approach to be implemented at a large scale level since it does not require any additional energy and it has proved to be efficient in plasmid amplification.

Keywords
Plasmid DNA; Amino acid limitation; Temperature up-shift; Flow cytometry; Real-time qPCR
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Impact of plasmid induction strategy on overall plasmid DNA yield and E. coli physiology using flow cytometry and real-time PCR
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 46, Issue 1, January 2011, Pages 174–181
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us