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Selected Publication:

Striedner, G; Pfaffenzeller, I; Markus, L; Nemecek, S; Grabherr, R; Bayer, K; .
(2010): Plasmid-free T7-based Escherichia coli expression systems.
Biotechnol Bioeng. 2010; 105(4):786-794 FullText FullText_BOKU

Abstract:
In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional PET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. Biotechnol. Bioeng. 2010;105: 786-794. (C) 2009 Wiley Periodicals, Inc.
Authors BOKU Wien:
Bayer Karl
Grabherr Reingard
Luchner Markus
Striedner Gerald
BOKU Gendermonitor:

Find related publications in this database (using NML MeSH Indexing)
Bacteriophage T7/genetics*;DNA-Directed RNA Polymerases/genetics;DNA-Directed RNA Polymerases/metabolism*;Escherichia coli/genetics*;Escherichia coli/metabolism;Gene Expression Regulation, Bacterial*;Genetic Vectors/genetics;Protein Engineering/methods*;Recombinant Proteins/biosynthesis*;Viral Proteins/genetics;Viral Proteins/metabolism*;

Find related publications in this database (Keywords)
plasmid-free
metabolic burden
transcription tuning
fed-batch


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