Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.
BACKGROUND
Recombinant protein production in Escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this process. Lately, qPCR is becoming the method of choice for this purpose. In the presented work, an improved qPCR method adopted for PCN determination in various fermentation processes was developed.


RESULTS
To avoid experimental errors arising from irreproducible DNA isolation, whole cells, treated by heating at 95 degrees C for 10 minutes prior to storage at -20 degrees C, were used as a template source. Relative quantification, taking into account different amplification efficiencies of amplicons for chromosome and plasmid, was used in the PCN calculation. The best reproducibility was achieved when the efficiency estimated for specific amplicon, obtained within one run, was averaged. It was demonstrated that the quantification range of 2 log units (100 to 10000 bacteria per well) enable quantification in each time point during fermentation. The method was applied to study PCN variation in fermentation at 25 degrees C and the correlation between PCN and protein accumulation was established.


CONCLUSIONS
Using whole cells as a template source and relative quantification considering different PCR amplification efficiencies are significant improvements of the qPCR method for PCN determination. Due to the approaches used, the method is suitable for PCN determination in fermentation processes using various media and conditions.
Skulj
Mihaela
M
Lek Pharmaceuticals d,d,, a Sandoz company, Verovskova 57, SI-1526 Ljubljana, Slovenia. mihaela.skulj@sandoz.com.
Okrslar
Veronika
V
Jalen
Spela
S
Jevsevar
Simona
S
Slanc
Petra
P
Strukelj
Borut
B
Menart
Viktor
V