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Current Microbiology

, Volume 20, Issue 6, pp 373–379 | Cite as

Stabilization of a plasmid-encoded LacZ phenotype inBacillus subtilis

  • Yuval Shoham
  • Arnold L. Demain
Article

Abstract

Recombinant plasmid pCED3 was structurally unstable inBacillus subtilis cultures grown in the presence of kanamycin to eliminate the effects of segregational instability. Analysis of 96 modified plasmids indicated that deletions in the plasmid occur at many different sites. The presence of plasmid pCED3 slowed the growth rate of theB. subtilis host. Cells that contained modified plasmids grew faster than the parental cells and took over the population. Two different methodologies were developed to reduce the cultural instability of the plasmid-directed LacZ+ phenotype. By growing the cells in a medium that supports a low growth rate, the growth rate ratio between modified and parental cells was reduced, resulting in a partial stabilization (40 generations) of the LacZ+ phenotype in the population [35]. Removal of a 4.77 kbEcoRI fragment (which consists primarily of the pBR322 replicon) from plasmid pCED3 produced a more stable plasmid derivative, designated pYS1. Cells harboring plasmid pYS1 grew faster than pCED3-bearing cells, although the level of activity of β-galactosidase was similar in both strains. By combining the two approaches (i.e., growth of pYS1-bearing cells in a medium that supports low growth rate), the LacZ+ phenotype was stably maintained in the cell population for over 170 generations. Under these conditions, there was no detectable difference between the growth rates of cells bearing the pYS1 plasmid and further modified plasmids.

Keywords

Growth Rate Cell Population Kanamycin Recombinant Plasmid Rate Ratio 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag New York Inc. 1990

Authors and Affiliations

  • Yuval Shoham
    • 1
  • Arnold L. Demain
    • 1
  1. 1.Fermentation Microbiology Laboratory, Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA

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