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

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...

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Autores principales: Freudenau, Inga, Lutter, Petra, Baier, Ruth, Schleef, Martin, Bednarz, Hanna, Lara, Alvaro R., Niehaus, Karsten
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555960/
https://www.ncbi.nlm.nih.gov/pubmed/26389114
http://dx.doi.org/10.3389/fbioe.2015.00127
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author Freudenau, Inga
Lutter, Petra
Baier, Ruth
Schleef, Martin
Bednarz, Hanna
Lara, Alvaro R.
Niehaus, Karsten
author_facet Freudenau, Inga
Lutter, Petra
Baier, Ruth
Schleef, Martin
Bednarz, Hanna
Lara, Alvaro R.
Niehaus, Karsten
author_sort Freudenau, Inga
collection PubMed
description 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.
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spelling pubmed-45559602015-09-18 ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation Freudenau, Inga Lutter, Petra Baier, Ruth Schleef, Martin Bednarz, Hanna Lara, Alvaro R. Niehaus, Karsten Front Bioeng Biotechnol Bioengineering and Biotechnology 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. Frontiers Media S.A. 2015-09-01 /pmc/articles/PMC4555960/ /pubmed/26389114 http://dx.doi.org/10.3389/fbioe.2015.00127 Text en Copyright © 2015 Freudenau, Lutter, Baier, Schleef, Bednarz, Lara and Niehaus. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Freudenau, Inga
Lutter, Petra
Baier, Ruth
Schleef, Martin
Bednarz, Hanna
Lara, Alvaro R.
Niehaus, Karsten
ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation
title ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation
title_full ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation
title_fullStr ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation
title_full_unstemmed ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation
title_short ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation
title_sort cole1-plasmid production in escherichia coli: mathematical simulation and experimental validation
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555960/
https://www.ncbi.nlm.nih.gov/pubmed/26389114
http://dx.doi.org/10.3389/fbioe.2015.00127
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