Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli
Open accessAbstract
BACKGROUND: Segregation of expression plasmids leads to loss of recombinant DNA from transformed bacterial cells due to the irregular distribution of plasmids between the daughter cells during cell division. Under non-selective conditions this segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells thus decreasing the yield of recombinant protein. Amongst the factors affecting segregational plasmid instability are: the plasmid design, plasmid copy-number, host cell genotype, fermentation conditions etc. This study aims to investigate the influence of transcription and translation on the segregation of recombinant plasmids designed for constitutive gene expression in Escherichia coli LE392 at glucose-limited continuous cultivation. To this end a series of pBR322-based plasmids carrying a synthetic human interferon-gamma (hIFNγ) gene placed under the control of different regulatory elements (promoter and ribosome-binding sites) were used as a model. RESULTS: Bacterial growth and product formation kinetics of transformed E. coli LE392 cells cultivated continuously were described by a structured kinetic model proposed by Lee et al. (1985). The obtained results demonstrated that both transcription and translation efficiency strongly affected plasmid segregation. The segregation of plasmid having a deleted promoter did not exceed 5% after 190 h of cultivation. The observed high plasmid stability was not related with an increase in the plasmid copy-number. A reverse correlation between the yield of recombinant protein (as modulated by using different ribosome binding sites) and segregational plasmid stability (determined by the above model) was also observed. CONCLUSIONS: Switching-off transcription of the hIFNγ gene has a stabilising effect on ColE1-like plasmids against segregation, which is not associated with an increase in the plasmid copy-number. The increased constitutive gene expression has a negative effect on segregational plasmid stability. A kinetic model proposed by Lee et al. (1985) was appropriate for description of E. coli cell growth and recombinant product formation in chemostat cultivations.
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Plasmid-based expression systems exhibit drawbacks including continuous amplification of plasmid copy number in prolonged cultivations and loss of plasmids over time, propagating generation of a plasmid-free subpopulation during induction.
“In general, the plasmid-based expression systems exhibit some drawbacks, including the continuous amplification of the plasmid copy number in prolonged cultivations and loss of plasmids over time, propagating the generation of a plasmid-free subpopulation during induction [73].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [73]
The Background and Results and Discussion (Plasmid segregation; page not provided) support plasmid loss during cultivation and the resulting growth of a plasmid-free subpopulation. They do not support continuous amplification of plasmid copy number: the paper describes a quasi steady-state copy number. The study used constitutive expression, so it also does not establish that this occurred during induction. (LLM, conf=0.88)
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- Authors
- Popov, M., Petrov, S., Nacheva, G., Ivanov, I., & Reichl, U.
- Affiliations
- Bulgarian Academy of Sciences (BG)
- Max Planck Institute for Dynamics of Complex Technical Systems (DE)
- Corresponding author
- Popov, Mladen
- Venue
- BMC Biotechnology
- Published
- 2011-03-01
- Volume
- 11
- Issue
- 1
- Pages
- 18-18
- ISSN
- 1472-6750 (ISSN-L)
- PMC
- PMC3061898
- OpenAlex
- W2010137391
- Cited by
- 38
- References
- 59
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 1 · 2022: 3 · 2021: 1 · 2020: 5 · 2019: 3 · 2018: 3 · 2017: 2 · 2015: 2 · 2014: 4 · 2013: 5 · 2012: 4
- FWCI
- 2.59
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- BMC Biotechnology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion, other-oa)
- Funders
- National Science Foundation
- European Commission
- Deutscher Akademischer Austauschdienst
- Fonds National de la Recherche Luxembourg
- Pohang University of Science and Technology
- Max Planck Institute for Dynamics of Complex Technical Systems Magdeburg
- European Social Fund
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Keywords
- plasmid segregation, recombinant gene expression, Escherichia coli, human interferon-gamma, promoter, ribosome binding site, transcription efficiency, translation efficiency, plasmid copy number, recombinant protein yield, chemostat cultivation
- MeSH terms
- DNA, Recombinant, Escherichia coli, Humans, Interferon-gamma, Plasmids, Recombinant Proteins, Gene Expression
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