Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli

Abstract

BackgroundSegregation 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.ResultsBacterial 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.ConclusionsSwitching-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.

Effect of the 3′-Terminal Truncation of the Human interferon-Gamma Gene on Plasmid Segregation inEscherichia Coli

“…This presents a clear evidence that inactivation of transcription has a strong stabilizing effect on the plasmid against segregation. This finding is supported also by a recent study where the role of transcription on plasmid segregation was investigated in a chemostat culture (18).…”

“…1. The plasmid pΔP 1 IFNγC was derived from the expression plasmid pIFNγC by removing the EcoRI/HindIII fragment (bearing both P 1 promoter and the SD sequence), blunting and ligation (18).…”

“…In a recent study (18) we have employed the mathematical models of Stewart and Levin (24) and Lee et al (13) to study the effect of transcription and translation on the segregation of pBR322-based plasmids expressing hIFNγ gene in E. coli LE392 cells under constitutive gene expression conditions. A reverse correlation between the yield of protein and plasmid stability was observed, which did not correlate with the variations in either plasmid copy-number or the specific bacterial growth rate.…”

Polar Fixation of Plasmids during Recombinant Protein Production in Bacillus megaterium Results in Population Heterogeneity

“…Recently, a relationship between gene expression and plasmid segregation was shown for ColE1-like plasmids in E. coli (37): it was argued that transcription and translation interfere with plasmid replication, leading to low copy numbers and finally to plasmid loss. We did not detect complete plasmid loss, and a highly related plasmid has been described as being stably maintained in B. megaterium, even without selective pressure (38). We eventually concluded that the transcriptional and translational machinery slows down the distribution of plasmids in B. megaterium.…”

Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

“…Conventionally this is achieved through cultivationbased methods in which diluted bacterial suspensions are spread on media with and without selection for plasmid-bearing cells or by replicating bacterial colonies (e.g., 50 or 100) from nonselective plates onto plasmid-selective and nonselective agar plates (4,5,(12)(13)(14)(15)(16)(17). However, this is labor-intensive, requires colony growth on agar, and has a built-in lag period of one or more days prior to knowing the results.…”