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ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation.

Abstract

Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production.

Authors+Show Affiliations

Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld , Bielefeld , Germany.Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld , Bielefeld , Germany.PlasmidFactory GmbH & Co. KG , Bielefeld , Germany.PlasmidFactory GmbH & Co. KG , Bielefeld , Germany.Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld , Bielefeld , Germany ; Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie (CeBiTec), Universität Bielefeld , Bielefeld , Germany.Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa , Mexico City , Mexico.Abteilung für Proteom- und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld , Bielefeld , Germany ; Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie (CeBiTec), Universität Bielefeld , Bielefeld , Germany.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

26389114

Citation

Freudenau, Inga, et al. "ColE1-Plasmid Production in Escherichia Coli: Mathematical Simulation and Experimental Validation." Frontiers in Bioengineering and Biotechnology, vol. 3, 2015, p. 127.
Freudenau I, Lutter P, Baier R, et al. ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation. Front Bioeng Biotechnol. 2015;3:127.
Freudenau, I., Lutter, P., Baier, R., Schleef, M., Bednarz, H., Lara, A. R., & Niehaus, K. (2015). ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation. Frontiers in Bioengineering and Biotechnology, 3, p. 127. doi:10.3389/fbioe.2015.00127.
Freudenau I, et al. ColE1-Plasmid Production in Escherichia Coli: Mathematical Simulation and Experimental Validation. Front Bioeng Biotechnol. 2015;3:127. PubMed PMID: 26389114.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation. AU - Freudenau,Inga, AU - Lutter,Petra, AU - Baier,Ruth, AU - Schleef,Martin, AU - Bednarz,Hanna, AU - Lara,Alvaro R, AU - Niehaus,Karsten, Y1 - 2015/09/01/ PY - 2015/06/05/received PY - 2015/08/13/accepted PY - 2015/9/22/entrez PY - 2015/9/22/pubmed PY - 2015/9/22/medline KW - biotechnology KW - high copy plasmid KW - modeling KW - ordinary differential equations KW - plasmid replication KW - small RNA KW - uncharged tRNA SP - 127 EP - 127 JF - Frontiers in bioengineering and biotechnology JO - Front Bioeng Biotechnol VL - 3 N2 - Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production. SN - 2296-4185 UR - https://www.unboundmedicine.com/medline/citation/26389114/ColE1_Plasmid_Production_in_Escherichia_coli:_Mathematical_Simulation_and_Experimental_Validation_ L2 - https://doi.org/10.3389/fbioe.2015.00127 DB - PRIME DP - Unbound Medicine ER -