title: Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli.
abstract: 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.
authors.affiliation: Institute of Molecular Biology Roumen Tsanev, Bulgarian Academy of Sciences, Acad, G, Bonchev Str,, 21, 1113 Sofia, Bulgaria. mnpopov@abv.bg
authors.forename: Mladen
authors.initials: M
authors.lastname: Popov
authors.forename: Stefan
authors.initials: S
authors.lastname: Petrov
authors.forename: Genoveva
authors.initials: G
authors.lastname: Nacheva
authors.forename: Ivan
authors.initials: I
authors.lastname: Ivanov
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
mesh_terms: D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D007371:Interferon-gamma