Determination of Plasmid Segregational Stability in a Growing Bacterial Population

  • M. Gabriela KramerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1409)


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 words

Plasmid 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)).


  1. 1.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory press, New York. ISBN 0879695773Google Scholar
  2. 2.
    Kramer MG, Khan SA, Espinosa M (1997) Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis. EMBO J 16:5784–5795PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Jensen RB, Dam M, Gerdes K (1994) Partitioning of plasmid R1. The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element. J Mol Biol 236:1299–1309CrossRefPubMedGoogle Scholar
  4. 4.
    Sobecky PA, Easter CL, Bear PD, Helinski DR (1996) Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2. J Bacteriol 178:2086–2093PubMedCentralPubMedGoogle Scholar
  5. 5.
    Diago-Navarro E, Hernandez-Arriaga AM, López-Villarejo J, Muñoz-Gómez AJ, Kamphuis MB, Boelens R, Lemonnier M, Díaz-Orejas R (2010) parD toxin-antitoxin system of plasmid R1--basic contributions, biotechnological applications and relationships with closely-related toxin-antitoxin systems. FEBS J 277:3097–3117CrossRefPubMedGoogle Scholar
  6. 6.
    Summers D (1998) Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability. Mol Microbiol 29:1137–1145CrossRefPubMedGoogle Scholar
  7. 7.
    del Solar G, Kramer G, Ballester S, Espinosa M (1993) Replication of the promiscuous plasmid pLS1: a region encompassing the minus origin of replication is associated with stable plasmid inheritance. Mol Gen Genet 241:97–105CrossRefPubMedGoogle Scholar
  8. 8.
    Kramer MG, Espinosa M, Misra TK, Khan SA (1999) Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids. Mol Microbiol 33:466–475CrossRefPubMedGoogle Scholar
  9. 9.
    Kramer MG, Espinosa M, Misra TK, Khan SA (1998) Lagging strand replication of rolling-circle plasmids: specific recognition of the ssoA-type origins in different gram-positive bacteria. Proc Natl Acad Sci U S A 95:10505–10510PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Field CM, Summers DK (2011) Multicopy plasmid stability: revisiting the dimer catastrophe. J Theor Biol 291:119–127CrossRefPubMedGoogle Scholar
  11. 11.
    Dunstan SJ, Simmons CP, Strugnell RA (2003) In vitro and in vivo stability of recombinant plasmids in a vaccine strain of Salmonella enterica var. Typhimurium. FEMS Immunol Med Microbiol 37:111–119CrossRefPubMedGoogle Scholar
  12. 12.
    Bauer H, Darji A, Chakraborty T, Weiss S (2005) Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids. Gene Ther 12:364–372CrossRefPubMedGoogle Scholar
  13. 13.
    Gahan ME, Webster DE, Wesselingh SL, Strugnell RA (2007) Impact of plasmid stability on oral DNA delivery by Salmonella enterica serovar Typhimurium. Vaccine 25:1476–1483CrossRefPubMedGoogle Scholar
  14. 14.
    Moreno M, Kramer MG, Yim L, Chabalgoity JA (2010) Salmonella as live Trojan horse for vaccine development and cancer gene therapy. Curr Gene Ther 10:56–76CrossRefPubMedGoogle Scholar
  15. 15.
    Kramer MG (2003) Orígenes de replicación de la cadena retrasada del plásmido pMV158 (Lagging-strand replication origins of plasmid pMV158). Universidad Complutense de Madrid (ed). ISBN: 9788466915298Google Scholar
  16. 16.
    Gonzalez R, Chabalgoity JA, and Kramer MG, unpublished resultsGoogle Scholar
  17. 17.
    Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedCentralPubMedGoogle Scholar
  18. 18.
    Luria SE, Burrous JW (1957) Hybridization between Escherichia coli and Shigella. J Bacteriol 74:461–476PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biotechnology, Instituto de Higiene, Facultad de MedicinaUniversidad de la RepúblicaMontevideoUruguay

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