Flow-cytometric detection of changes in the physiological state ofE. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation
| Autor: | Michael Berney, Markus Keller, Thomas Egli, Verena Looser, Frederik Hammes, Karin Kovar |
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| Rok vydání: | 2005 |
| Předmět: |
Time Factors
Acetates medicine.disease_cause Applied Microbiology and Biotechnology law.invention Cell membrane chemistry.chemical_compound Bioreactors law Ethidium Flow cytometry education.field_of_study Ethidium bromide Escherichia coli Proteins Physiological state Recombinant Proteins Fedbatch Cell biology medicine.anatomical_structure Biochemistry Oxygenases Recombinant DNA Propidium Biotechnology Xylene monooxygenase Propidium iodide Population Heterologous 660.6: Biotechnologie Bioengineering Biology Industrial Microbiology Recombinant membrane protein Escherichia coli medicine education Cell damage Fluorescent Dyes Cell Membrane E. coli Membrane Proteins medicine.disease Carbon Culture Media Glucose Microscopy Fluorescence chemistry Membrane protein Fermentation |
| Zdroj: | Biotechnology and Bioengineering. 92:69-78 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.20575 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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