Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation
Binder D, Probst C, Grünberger A, Hilgers F, Loeschcke A, Jaeger K-E, Kohlheyer D, Drepper T (2016)
PLoS one 11(8): e0160711.
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| Veröffentlicht | Englisch
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Autor*in
Binder, Dennis;
Probst, Christopher;
Grünberger, AlexanderUniBi;
Hilgers, Fabienne;
Loeschcke, Anita;
Jaeger, Karl-Erich;
Kohlheyer, Dietrich;
Drepper, Thomas
Abstract / Bemerkung
Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust triggering of bacterial gene expression in E. coli in a homogeneous fashion.
Erscheinungsjahr
2016
Zeitschriftentitel
PLoS one
Band
11
Ausgabe
8
Art.-Nr.
e0160711
Urheberrecht / Lizenzen
ISBN
1932-6203
ISSN
1932-6203
Page URI
https://pub.uni-bielefeld.de/record/2912514
Zitieren
Binder D, Probst C, Grünberger A, et al. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one. 2016;11(8): e0160711.
Binder, D., Probst, C., Grünberger, A., Hilgers, F., Loeschcke, A., Jaeger, K. - E., Kohlheyer, D., et al. (2016). Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one, 11(8), e0160711. doi:10.1371/journal.pone.0160711
Binder, Dennis, Probst, Christopher, Grünberger, Alexander, Hilgers, Fabienne, Loeschcke, Anita, Jaeger, Karl-Erich, Kohlheyer, Dietrich, and Drepper, Thomas. 2016. “Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation”. PLoS one 11 (8): e0160711.
Binder, D., Probst, C., Grünberger, A., Hilgers, F., Loeschcke, A., Jaeger, K. - E., Kohlheyer, D., and Drepper, T. (2016). Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one 11:e0160711.
Binder, D., et al., 2016. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one, 11(8): e0160711.
D. Binder, et al., “Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation”, PLoS one, vol. 11, 2016, : e0160711.
Binder, D., Probst, C., Grünberger, A., Hilgers, F., Loeschcke, A., Jaeger, K.-E., Kohlheyer, D., Drepper, T.: Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one. 11, : e0160711 (2016).
Binder, Dennis, Probst, Christopher, Grünberger, Alexander, Hilgers, Fabienne, Loeschcke, Anita, Jaeger, Karl-Erich, Kohlheyer, Dietrich, and Drepper, Thomas. “Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation”. PLoS one 11.8 (2016): e0160711.
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2019-09-06T09:18:49Z
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Daten bereitgestellt von European Bioinformatics Institute (EBI)
7 Zitationen in Europe PMC
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Lee SK, Chou HH, Pfleger BF, Newman JD, Yoshikuni Y, Keasling JD., Appl. Environ. Microbiol. 73(18), 2007
PMID: 17644634
Charting microbial phenotypes in multiplex nanoliter batch bioreactors.
Dai J, Yoon SH, Sim HY, Yang YS, Oh TK, Kim JF, Hong JW., Anal. Chem. 85(12), 2013
PMID: 23581968
Dai J, Yoon SH, Sim HY, Yang YS, Oh TK, Kim JF, Hong JW., Anal. Chem. 85(12), 2013
PMID: 23581968
Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch
AUTHOR UNKNOWN, 2010
AUTHOR UNKNOWN, 2010
Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers.
Khlebnikov A, Keasling JD., Biotechnol. Prog. 18(3), 2002
PMID: 12052093
Khlebnikov A, Keasling JD., Biotechnol. Prog. 18(3), 2002
PMID: 12052093
Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture.
Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD., J. Bacteriol. 182(24), 2000
PMID: 11092865
Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD., J. Bacteriol. 182(24), 2000
PMID: 11092865
Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.
Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, Keasling JD., Microbiology (Reading, Engl.) 147(Pt 12), 2001
PMID: 11739756
Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, Keasling JD., Microbiology (Reading, Engl.) 147(Pt 12), 2001
PMID: 11739756
Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity.
Morgan-Kiss RM, Wadler C, Cronan JE Jr., Proc. Natl. Acad. Sci. U.S.A. 99(11), 2002
PMID: 12032290
Morgan-Kiss RM, Wadler C, Cronan JE Jr., Proc. Natl. Acad. Sci. U.S.A. 99(11), 2002
PMID: 12032290
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