Study of plasmid-based expression level heterogeneity under plasmid-curing like conditions in Cupriavidus necator.

Autor: Boy C; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Lesage J; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Alfenore S; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Guillouet SE; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France., Gorret N; TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France. Electronic address: ngorret@insa-toulouse.fr.
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 2022 Feb 10; Vol. 345, pp. 17-29. Date of Electronic Publication: 2022 Jan 05.
DOI: 10.1016/j.jbiotec.2021.12.015
Abstrakt: Plasmid expression level heterogeneity in Cupriavidus necator was studied in response to stringent culture conditions, supposed to enhance plasmid instability, through plasmid curing strategies. Two plasmid curing strategies were compared based on their efficiency at generating heterogeneity in batch: rifampicin addition and temperature increase. A temperature increase from 30° to 37 °C was the most efficient plasmid curing strategy. To generate a heterogeneous population in terms of plasmid expression levels, successive batches at supra-optimal culture temperature (i.e. 37 °C) were initially conducted. Three distinct fluorescent subpopulations P 0 (not fluorescent), P 1 (low fluorescence intensity, median = 1 10 3 ) and P 2 (high fluorescence intensity, median = 6 10 3 ) were obtained. From there, the chemostat culture was implemented to study the long-term stress response under well-controlled environment at defined dilution rates. For dilution rates comprised between 0.05 and 0.10 h -1 , the subpopulation P 2 (62% vs 90%) was favored compared to P 1 cells (54% vs 1%), especially when growth rate increased. Our biosensor was efficient at discriminating subpopulation presenting different expression levels under stringent culture conditions. Plus, we showed that controlling growth kinetics had a stabilizing impact on plasmid expression levels, even under heterogeneous expression conditions.
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Databáze: MEDLINE
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