Determination of Plasmid Segregational Stability in a Growing Bacterial Population
Bacterial plasmids are extensively used as cloning vectors for a number of genes for academic and commercial purposes. Moreover, attenuated bacteria carrying recombinant plasmids expressing genes with anti-tumor activity have shown promising therapeutic results in animal models of cancer. Equitable plasmid distribution between daughter cells during cell division, i.e., plasmid segregational stability, depends on many factors, including the plasmid copy number, its replication mechanism, the levels of recombinant gene expression, the type of bacterial host, and the metabolic burden associated with all these factors. Plasmid vectors usually code for antibiotic-resistant functions, and, in order to enrich the culture with bacteria containing plasmids, antibiotic selective pressure is commonly used to eliminate plasmid-free segregants from the growing population. However, administration of antibiotics can be inconvenient for many industrial and therapeutic applications. Extensive ongoing research is being carried out to develop stably-inherited plasmid vectors. Here, I present an easy and precise method for determining the kinetics of plasmid loss or maintenance for every ten generations of bacterial growth in culture.
Key wordsPlasmid Stability Cell division Bacterial population
I would like to thank Dr. Gloria del Solar and Dr. Manuel Espinosa (Center for Biological Research, Consejo Superior de Investigaciones Cientificas, Madrid, Spain) for having taught me this method in the early 90s. Thanks also to Dr. Jose A. Chabalgoity (Department of Biochemistry, School of Medicine, Universidad de la República, Montevideo, Uruguay) for his encouragement to write this chapter and to our undergraduate student, Rodrigo Gonzalez, for having tested this plasmid stability method in several Gram (−) host strains (Grant support: Comision Honoraria de Lucha Contra el Cáncer (CHLCC-Kramer 2009)).
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