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

ABSTRACT

Segregation of plasmids with relaxed control of replication is due to their non-random distribution between the daughter cells during cell division. Plasmid segregation is a biological phenomenon influenced by many factors like type of replicon, presence of partitioning elements, plasmid copy-number, cultivation conditions, host genotype, etc.

The aim of this study was to investigate the effect of systematic changes in the human interferon-gamma (hIFNγ) gene on the segregation of expression plasmids in E. coli cells. To this end a series of 3'-truncated hIFNγ genes were cloned in a pBR322-based expression plasmid containing a strong constitutive promoter (T5P25) and a synthetic ribosome binding site. The series of plasmids thus constructed was transformed in E. coli LE392 cells and their segregation was investigated under batch fermentation conditions in non-selective Luria-Bertani medium. To describe the population dynamics, a mathematical model proposed by Stewart and Levin was applied. Using nonlinear fitting technique, the difference in specific growth rate between plasmid-free and plasmid-harbouring cells and the specific generation rate of plasmid-free cells (as well as the relative plasmid loss rate) were determined.

The obtained results demonstrated that small changes in the 3'-terminus of the hIFNγ gene strongly affect plasmid segregation. To explain this influence the model parameters were compared with experimental data characterizing hIFNγ gene expression such as yield of recombinant protein, hIFNγ-mRNA and plasmid copy-number. A clear correlation was found between the relative plasmid loss rate and hIFNγ-mRNA content, which however was not associated with the plasmid copy-number value.

Keywords:

• Escherichia coli
• human interferon-gamma
• hIFNγ
• Stewart and Levin model
• plasmid segregation
• plasmid instability