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Advisor(s)
Abstract(s)
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.
Description
Keywords
Plasmid DNA Amino acid limitation Temperature up-shift Flow cytometry Real-time qPCR