Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation

Looser, Verena; Hammes, Frederik; Keller, Markus; Berney, Michael; Kovar, Karin; Egli, Thomas

doi:10.1002/bit.20575

Full metadata record

DC Field	Value	Language
dc.contributor.author	Looser, Verena	-
dc.contributor.author	Hammes, Frederik	-
dc.contributor.author	Keller, Markus	-
dc.contributor.author	Berney, Michael	-
dc.contributor.author	Kovar, Karin	-
dc.contributor.author	Egli, Thomas	-
dc.date.accessioned	2018-10-08T11:43:07Z	-
dc.date.available	2018-10-08T11:43:07Z	-
dc.date.issued	2005-10-05	-
dc.identifier.issn	1097-0290	de_CH
dc.identifier.issn	0006-3592	de_CH
dc.identifier.uri	https://digitalcollection.zhaw.ch/handle/11475/11467	-
dc.description.abstract	The key to optimizing productivity during industrial fermentations is the ability to rapidly monitor and interpret the physiological state of single microbial cells in a population and to recognize and characterize different sub-populations. Here, a flow cytometry-based method for the reproducible detection of changes in membrane function and/or structure of recombinant E. coli JM101 (pSPZ3) expressing xylene monooxygenase (XMO), was developed. XMO expression led to compromised but not permeabilized cell membranes. This was deduced from the fact that recombinant cells only stained with ethidium bromide (EB) and not with propidium iodide (PI). During the glucose-limited fedbatch cultivation, an increase from 25% to 95% of EB-stained cells was observed, occurring between 2 and 5 h after induction. Control experiments confirmed that this increase was due to the recombinant protein production and not caused by any possible effects of varying substrate availability, high cell density, plasmid replication or the presence of the inducing agent. We hypothesize that the integration of the recombinant protein into the cell membrane physically disrupted the functionality of the efflux pumps, thus resulting in EB-staining of the recombinant cells. This method enabled us to detect changes in the physiological state of single cells 2-4 h before other indications of partial cell damage, such as unbalanced growth, acetate accumulation and an increased CO(2) production rate, were observed. This method therefore shows promise with respect to the further development of an early-warning system to prevent sudden productivity decreases in processes with recombinant E. coli expressing heterologous membrane proteins.	de_CH
dc.language.iso	en	de_CH
dc.publisher	Wiley	de_CH
dc.relation.ispartof	Biotechnology & Bioengineering	de_CH
dc.rights	Licence according to publishing contract	de_CH
dc.subject	Ethidium bromide	de_CH
dc.subject	Fedbatch	de_CH
dc.subject	E. coli	de_CH
dc.subject	Flow cytometry	de_CH
dc.subject	Physiological state	de_CH
dc.subject	Propidium iodide	de_CH
dc.subject	Recombinant membrane protein	de_CH
dc.subject	Xylene monooxygenase	de_CH
dc.subject.ddc	660.6: Biotechnologie	de_CH
dc.title	Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation	de_CH
dc.type	Beitrag in wissenschaftlicher Zeitschrift	de_CH
dcterms.type	Text	de_CH
zhaw.departement	Life Sciences und Facility Management	de_CH
zhaw.organisationalunit	Institut für Chemie und Biotechnologie (ICBT)	de_CH
dc.identifier.doi	10.1002/bit.20575	de_CH
zhaw.funding.eu	No	de_CH
zhaw.issue	1	de_CH
zhaw.originated.zhaw	Yes	de_CH
zhaw.pages.end	78	de_CH
zhaw.pages.start	69	de_CH
zhaw.publication.status	publishedVersion	de_CH
zhaw.volume	92	de_CH
zhaw.publication.review	Peer review (Publikation)	de_CH
zhaw.webfeed	Biokatalyse	de_CH
Appears in collections:	Publikationen Life Sciences und Facility Management

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Looser, V., Hammes, F., Keller, M., Berney, M., Kovar, K., & Egli, T. (2005). Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation. Biotechnology & Bioengineering, 92(1), 69–78. https://doi.org/10.1002/bit.20575

Looser, V. et al. (2005) ‘Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation’, Biotechnology & Bioengineering, 92(1), pp. 69–78. Available at: https://doi.org/10.1002/bit.20575.

V. Looser, F. Hammes, M. Keller, M. Berney, K. Kovar, and T. Egli, “Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation,” Biotechnology & Bioengineering, vol. 92, no. 1, pp. 69–78, Oct. 2005, doi: 10.1002/bit.20575.

LOOSER, Verena, Frederik HAMMES, Markus KELLER, Michael BERNEY, Karin KOVAR und Thomas EGLI, 2005. Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation. Biotechnology & Bioengineering. 5 Oktober 2005. Bd. 92, Nr. 1, S. 69–78. DOI 10.1002/bit.20575

Looser, Verena, Frederik Hammes, Markus Keller, Michael Berney, Karin Kovar, and Thomas Egli. 2005. “Flow-Cytometric Detection of Changes in the Physiological State of E. Coli Expressing a Heterologous Membrane Protein during Carbon-Limited Fedbatch Cultivation.” Biotechnology & Bioengineering 92 (1): 69–78. https://doi.org/10.1002/bit.20575.

Looser, Verena, et al. “Flow-Cytometric Detection of Changes in the Physiological State of E. Coli Expressing a Heterologous Membrane Protein during Carbon-Limited Fedbatch Cultivation.” Biotechnology & Bioengineering, vol. 92, no. 1, Oct. 2005, pp. 69–78, https://doi.org/10.1002/bit.20575.