An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics

Kimberly L. Ogden, Robert H. Davis, Autin L. Taylor

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth‐rate differential between plasmid‐bearing and plasmid‐free cells, and thereby affects the culture dynamics of a plasmid‐containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl tranferase (CAT). Experimental and theoretical results show that plasmid‐bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild‐type levels, and thus have more of a metabolic burden than do the plasmid‐bearng cells that overproduce CAT. However, when the level of the plasmid‐directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid‐bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid‐containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments. © 1992 John Wiley & Sons, Inc.

Original languageEnglish (US)
Pages (from-to)1027-1038
Number of pages12
JournalBiotechnology and Bioengineering
Volume40
Issue number9
DOIs
StatePublished - Nov 1992

Keywords

  • continuous fermentation
  • gene expression
  • pili
  • plasmid segregation

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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