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Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

Skulj M, Okrslar V, Jalen S, Jevsevar S, Slanc P, Strukelj B, Menart V - Microb. Cell Fact. (2008)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lek Pharmaceuticals d,d,, a Sandoz company, Verovskova 57, SI-1526 Ljubljana, Slovenia. mihaela.skulj@sandoz.com.

ABSTRACT

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Accumulation level of hG-CSF in fermentations at 25°C without ampicillin (a) and with ampicillin (b). Escherichia coli BL21 (DE3) strain transformed with pET3a plasmid with gene for hG-CSF was used. Samples were taken every four hours, bands corresponding to hG-CSF are framed. M stands for Molecular weight standard Mark 12 with (Invitrogen) with purified hG-CSF; S stands for standard, purified hG-CSF.
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Figure 1: Accumulation level of hG-CSF in fermentations at 25°C without ampicillin (a) and with ampicillin (b). Escherichia coli BL21 (DE3) strain transformed with pET3a plasmid with gene for hG-CSF was used. Samples were taken every four hours, bands corresponding to hG-CSF are framed. M stands for Molecular weight standard Mark 12 with (Invitrogen) with purified hG-CSF; S stands for standard, purified hG-CSF.

Mentions: Samples for SDS-PAGE analysis were taken every 2 hours. SDS-PAGE for protein detection was performed on 4–12% gradient gels using the NuPage Novex Bis-Tris SDS-PAGE (Invitrogen) system. SDS-PAGE gels were stained by SimplyBlue Safe Stain (Invitrogen) (Fig. 1). The accumulation level of hG-CSF in total cellular proteins was determined by densitometric analysis of SDS-PAGE gels stained by Colloidal Blue Stain Kit (Invitrogen) which enables quantification due to linear response of the proteins ranging from 0.1 – 2 μg. Densitometer model ProExpress Imaging System (Perkin Elmer) and TotalLab100 version 2006 software were used for the quantification of the accumulation level of hG-CSF.


Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes.

Skulj M, Okrslar V, Jalen S, Jevsevar S, Slanc P, Strukelj B, Menart V - Microb. Cell Fact. (2008)

Accumulation level of hG-CSF in fermentations at 25°C without ampicillin (a) and with ampicillin (b). Escherichia coli BL21 (DE3) strain transformed with pET3a plasmid with gene for hG-CSF was used. Samples were taken every four hours, bands corresponding to hG-CSF are framed. M stands for Molecular weight standard Mark 12 with (Invitrogen) with purified hG-CSF; S stands for standard, purified hG-CSF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2311272&req=5

Figure 1: Accumulation level of hG-CSF in fermentations at 25°C without ampicillin (a) and with ampicillin (b). Escherichia coli BL21 (DE3) strain transformed with pET3a plasmid with gene for hG-CSF was used. Samples were taken every four hours, bands corresponding to hG-CSF are framed. M stands for Molecular weight standard Mark 12 with (Invitrogen) with purified hG-CSF; S stands for standard, purified hG-CSF.
Mentions: Samples for SDS-PAGE analysis were taken every 2 hours. SDS-PAGE for protein detection was performed on 4–12% gradient gels using the NuPage Novex Bis-Tris SDS-PAGE (Invitrogen) system. SDS-PAGE gels were stained by SimplyBlue Safe Stain (Invitrogen) (Fig. 1). The accumulation level of hG-CSF in total cellular proteins was determined by densitometric analysis of SDS-PAGE gels stained by Colloidal Blue Stain Kit (Invitrogen) which enables quantification due to linear response of the proteins ranging from 0.1 – 2 μg. Densitometer model ProExpress Imaging System (Perkin Elmer) and TotalLab100 version 2006 software were used for the quantification of the accumulation level of hG-CSF.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lek Pharmaceuticals d,d,, a Sandoz company, Verovskova 57, SI-1526 Ljubljana, Slovenia. mihaela.skulj@sandoz.com.

ABSTRACT

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus