Biotechnol. Prog. 1993, 9, 218-220
Plasmid Stability in Immobilized Mixed Cultures of Recombinant Escherichia coli
Vildan Dinqbag,+Amable Hortaqsu,**t and Agnes Camurdanz
Departments of Chemical Engineering and Biology, Boiazici University, Bebek, Istanbul 80815, Turkey
The growth behavior of a mixed culture of plasmid-free and plasmid-containing (YEp352) Escher :hia coli HBlOlcells in free suspension and immobilized conditions was studied experimentally. I t was found that immobilization in calcium alginate beads delayed the reduction of the plasmid-containing fraction for a period of time inversely related to the total number of cells initially immobilized. A simple unstructured model that assumes compartmentalization of cells in the immobilization matrix a t thebeginning of growth gave a fit which was in good agreement with the data.
Introduction A major problem in the industrial application of recombinant DNA technology is the instability of the recombinant plasmid, which tends to disappear from the culture over a long period of cultivation. This could be due to the loss of plasmid from some bacterial cells, on which a specific gene is cloned, andlor due tothe overgrowth of plasmid-free cells in the culture. Some strategies for improving the stability of recombinant plasmids are already known. However, most of these approaches involve manipulation of the plasmid or may not be favorable for industrial applications, such as the addition of antibiotics to fermentation media for selection of plasmid-containing cells (Lee at al., 1988). Immobilizationof the cells has been used as an experimental approach to enhance plasmid stability (Sayadi et al., 1989;Walls and Gainer, 1989; Oriel, 1988;Nasri et al., 1987). To understand the mechanism by which immobilization increases the plasmid stability, a mixed culture of plasmid-free and plasmid-containing (YEp352) Escherichia coli HBlOl cells were grown in freely suspended and in immobilized conditions.A kinetic model based on compartmentalized growth of the mixed population is used to explain the experimentally observed stability in the immobilized system. Materials and Methods Organisms. E. coli strain HBlOl with and without the plasmid vector YEp352 was kindly provided by Prof. Ferda Mavituna, University of Manchester. The host strain HBlOl is a hybrid K12/B strain (Wood and Peretti, 1990;Boyer and Roulland, 1969). Plasmid. The nonconjugative plasmid YEp352 has a size of 5.2 kb. The selective marker in E . coli is ampicillin resistance gene (ampR)and the selective marker is URA3 in yeast. The plasmid carries the promoter and lac-Za sites of the lac operon. The structural genes Z and a of the operon code for the amino acid sequences of 0-galactosidase and transacetylase,respectively. Culture Medium. The strains of E. coli were kept in gly2erol solution at -70 "C. M9 minimal medium is composed of, per liter of deionized and distilled water, 1 mL of 1 M MgS04.7H20, 1 mL of 1 M CaC12, 10 mL of
* Author to whom correspondence should be addressed.
Department of Chemical Engineering. Department of Biology.
20% glucose, 20 mg ofL-leucine, 20 mg of L-proline, 1mL of 1 M thiamine#HC1,6g of Na*HP04,3 g of KH2P04,l g of NH4Cl, and 0.5 g of NaC1. The pH was adjusted to 7.3 with 10 N NaOH. To increase the growth rate of the cells, double-supplemented minimal medium (M9+) has been used. Amounts of glucose and amino acids were doubled in this medium. LB medium was composed of 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaC1.To prepare Petri dishes containing solid medium, LB medium was supplemented with 15-20 g/L of agar (Difco). For detection of plasmidcontaining cells, 150 mg/mL ampicillin (Sigma) was added to the solid medium. Preparation of Alginate Gel Beads. The sodium salt of alginic acid has been used to prepare alginate gel beads. The method used was similar to the one described by Martinsen et al. (1989)....