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Effects of a Recombinant Gene Expression on ColE1-like Plasmid Segregation in Escherichia Coli

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Effects of a Recombinant Gene Expression on ColE1-like Plasmid Segregation in Escherichia Coli

Mladen Popov et al. BMC Biotechnol.

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.

Figures

Figure 1
Figure 1
Functional map of the plasmid pP1-(SD)-hIFNγ. P1 - strong constitutive promoter; SD - Shine & Dalgarno consensus sequence; hIFNγ - human interferon gamma gene coding for 143 amino acids; bla (ApR) - β-lactamase gene; tet*- truncated tetracycline resistance gene; rop - gene coding for the ROP protein (regulating plasmid copy-number); ori - origin of replication.
Figure 2
Figure 2
Plasmid-harbouring cell fraction z as a function of cultivation time. Experimental results were processed and simulations were carried out employing Eq. 23 and using the software product Berkeley Madonna, Version 8.3.9. (black squares and line - pP1-(SD)-hIFNγ; red squares and line - pP1-(4SD)-hIFNγ; blue squares and line - pP1-(ΔSD)-hIFNγ; green squares - pΔP1-(ΔSD)-hIFNγ. Data points and lines represent experimental results and trajectories predicted by the model calculations, respectively.)
Figure 3
Figure 3
Growth and plasmid loss kinetics of transformed E. coli LE392 cells in a chemostat culture. Experimental data (data points) and simulation curves obtained by the model of Lee et al. (lines): plasmid-harbouring biomass, x+ (blue squares and line); plasmid-free biomass, x- (blue triangles and line); total biomass, x (green squares and line); glucose concentration, s (green triangles and line); hIFNγ concentration, p (red squares and line) and plasmid-harbouring cell fraction, z (black squares and line) for cells carrying the plasmid pP1-(SD)-hIFNγ (Figure 3A), pP1-(4SD)-hIFNγ (Figure 3B) and pP1-(ΔSD)-hIFNγ (Figure 3C).
Figure 4
Figure 4
Simulated specific growth rate difference and specific growth rate of plasmid-harbouring cells. Specific growth rate difference Δ (solid lines) and specific growth rate of plasmid-harbouring cells μ+ (dashed lines) as a function of cultivation time predicted by the model of Lee et al. for the plasmids pP1-(SD)-hIFNγ (black lines), pP1-(4SD)-hIFNγ (red lines) and pP1-(ΔSD)-hIFNγ (blue lines).
Figure 5
Figure 5
Relationship between recombinant gene control elements and relative plasmid loss rate. Interconnections between the effects caused by the modifications of the recombinant gene control elements (A) and factors influencing the relative plasmid loss rate θ (B).
Figure 6
Figure 6
Specific rate of generation of plasmid-free cells Θ simulated by the model of Lee et al. The simulated graphs of Θ for pP1-(SD)-hIFNγ, pP1-(4SD)-hIFNγ and pP1-(ΔSD)-hIFNγ are presented with black, red and blue line, respectively.

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