Gezielte Optimierung von Escherichia coli BL21(DE3)

Noll, Stephan; Reyelt, Jan; Rysiok, Thomas; Kellner, Roland; Güssow, Detlef; Jäkel, Stefan; Hager, Stefanie; Kranz, Harald

doi:10.1007/s12268-013-0292-2

Cited by 2 publications

(6 citation statements)

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“…Apart from the marker for oxygen limitation in batch cultures, E. coli G5 T7E2 generally exhibited higher mean fluorescence values for all markers under all conditions investigated. At the same time, population variation respectively noise in gene expression was predominantly lower than for E. coli G7 BL21(DE3) which points towards higher alignment and robustness of E. coli G5 T7E2 cultures as also suspected earlier [45].…”

Section: Single Cell Level Physiologysupporting

confidence: 70%

“…Furthermore, the E. coli T7E2 based triple reporter strain showed a higher diversity in by-product formation in different phases of the batch as well as in response to the pulse, nevertheless this also resulted in lower steady state biomass concentrations in chemostat cultures at different dilution rates. As mentioned above, the reason could be a higher flexibility in the central carbon metabolism of E. coli G5 T7E2 [45]. Surprisingly, while E. coli G7 BL21(DE3) cometabolized acetate and glucose in batch and continuous culture, E. coli G5 T7E2 rather consumed acetate subsequently to glucose.…”

Section: Differences Between the Two Triple Reporter Strains Populatimentioning

confidence: 97%

“…Two Escherichia coli triple reporter strains, E. coli G7 BL21(DE3) and E. coli G5 T7E2 , expressing three chromosomally integrated fluorescent proteins connected to the expression of three different cellular markers (see below and Additional file 1: Table A1) were characterized in this study. E. coli G7 BL21(DE3) originates from wildtype E. coli BL21(DE3), whereas E. coli G5 T7E2 is based on a modified version of E. coli BL21(DE3), T7E2 [45]. Additionally, during initial characterization experiments the corresponding single-and double reporter strains were employed (see Additional file 1).…”

Section: Triple Reporter Strainsmentioning

confidence: 99%

“…E. coli G5 T7E2, and its wildtype however, behave similar but slightly different from the other two strains (see also Additional file 2). One reason could be, that E. coli T7E2 carries a knock-in of pgl that encodes for 6-phosphogluconolactonase and which E. coli BL21(DE3) lacks [45]. A loss of this gene leads to a blockage of the pentose-phosphate-pathway, so that intermediates of the remaining part of the central carbon metabolism can be stronger accumulated.…”

Section: Population Level Physiologymentioning

confidence: 99%

“…Escherichia coli BL21(DE3) has several advantageous characteristics like low acetate production at high glucose levels as well as being protease deficient [44]. The second strain used in this study, E. coli T7E2, should be more robust than its ancestor in an industrial cultivation setup [45]. Its modifications are related to removal of major parts of the lambda DE3 prophage and replacement of the cryptic prophage Rac by the pgl gene encoding 6-phosphogluconolactonase which BL21(DE3) has lost and that prevents accumulation of sugar-intermediates.…”

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confidence: 99%

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Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

et al. 2020

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Background: Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. Results: Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. Conclusions: Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.

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Section: Single Cell Level Physiologysupporting

confidence: 70%

Section: Differences Between the Two Triple Reporter Strains Populatimentioning

confidence: 97%

Section: Triple Reporter Strainsmentioning

confidence: 99%

Section: Population Level Physiologymentioning

confidence: 99%

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confidence: 99%

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Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

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Method for plasmid-based antibiotic-free fermentation

Brechun,

Förschle,

Schmidt

et al. 2024

Microb Cell Fact

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Background Antibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological manufacturing. Overuse of antibiotics and antibiotic resistance genes (ARG) contributes to the development of antimicrobial resistance, which is a growing threat to modern medicine. Antibiotics themselves are costly and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. Furthermore, constitutive expression of a plasmid-encoded antibiotic resistance gene imposes a significant metabolic burden on the cells. For many fermentation products (e.g., in nutrition and medicine), the use of antibiotic resistance genes is subject to strict regulations and should be avoided. We present a method for plasmid selection and maintenance with stringent selection pressure that is independent of antibiotics and ARG. Furthermore, it can be used without any restrictions regarding culture medium and temperature. Results The developed method involves modification of a bacterial strain such that an essential gene is expressed genomically under the control of an inducible promoter. A copy of the same essential gene with the endogenous promoter is supplied on a plasmid for selection. In the absence of the inducer for the genomic copy of the essential gene, cells rely on expression of the plasmid-encoded gene copy, leading to tight selection for plasmid maintenance. Induction of the genomic copy of the essential gene enables the engineered strain to be propagated in the absence of a plasmid. Here, we describe the genetic setup and demonstrate long-term, tight selection for plasmid maintenance with a variety of different plasmids and E. coli strains. Conclusions This method facilitates plasmid-based fermentations by eliminating the need for antibiotic selection and improving plasmid maintenance.

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Gezielte Optimierung von Escherichia coli BL21(DE3)

Cited by 2 publications

References 11 publications

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

Method for plasmid-based antibiotic-free fermentation

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