Molecular Biotechnology

, Volume 9, Issue 1, pp 17–24 | Cite as

Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes



We have developed a tightly controlled, two-stage expression system. It is based on a single plasmid that carries the TetR repressor/P tet promoter/O tet operator for the first-stage control, and the Flp recombinase/FRT sites for the second-stage control. The gene to be expressed (GENE) is cloned in an inverted orientation (with respect to the stationary promoter) into a multiple-cloning site (MCS) located between two convergentFRT1 andFRT2 sites. In the OFF stage, no inadvertent transcription can enter the 5′ end of clonedGENE because of fourrrnBT1 terminators, located just outside theFRT1-MCS-FRT2 cassette and because theFRT2 construct was deprived of any promoter function. When using thelacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stagetetR/P tetOtet-lacZ vectors. To enter the ON stage, thetetR/P tetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts theFRT1-MCS-FRT2 module together with the clonedGENE. This results in the massiveGENE expression from one (pInvMS) or two (pImpMS) stationary promoters.

Index Entries

Escherichia coli two-stage control tet promoter inversion cassette 


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  1. 1.
    Makrides, S. C. (1996) Strategies for achieving high-level expression of genes inEscherichia coli.Microbiol. Rev. 60, 512–538.Google Scholar
  2. 2.
    Hillen, W. and Berens, C. (1994) Mechanisms underlying expression of Tn10-encoded tetracycline resistance.Annu. Rev. Microbiol. 48, 345–369.PubMedCrossRefGoogle Scholar
  3. 3.
    Szybalski, W. (1993) From the double-helix to novel approaches to the sequencing of large genomes.Gene 135, 279–290.PubMedCrossRefGoogle Scholar
  4. 4.
    Pósfai, G., Koob, M., Hradečná, Z., Hasan, N., Filutowicz, M., and Szybalski, W. (1994)In vivo excision and amplification of large segments of theEscherichia coli genome.Nucleic Acids Res. 22, 2392–2398.PubMedCrossRefGoogle Scholar
  5. 5.
    Wild, J., Hradecná, Z., Pósfai, G., and Szybalski, W. (1996) A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing.Gene 179, 181–188.CrossRefGoogle Scholar
  6. 6.
    Podhajska, A. J., Hasan, N., and Szybalski, W. (1985) Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activatedatt-nutL-p-att-N module.Gene 40, 163–168.PubMedCrossRefGoogle Scholar
  7. 7.
    Hasan, N. and Szybalski, W. (1987) Control of cloned gene expression by promoter inversion in vivo: construction of improved vectors with a multiple cloning site and thep tae promoter.Gene 56, 145–151.PubMedCrossRefGoogle Scholar
  8. 8.
    Cox, M. M. (1988) FLP site-specific recombination system ofSaccharomyces cerevisiae, inGenetic Recombination vol. 1. (Kucherlapati, R. and Smith, G. R., eds.), American Society for Microbiology, Washington, DC, pp. 429–443.Google Scholar
  9. 9.
    Futcher, A. B. (1988) The 2 μm circle plasmid ofSaccharomyces cerevisiae.Yeast 4, 27–40.PubMedCrossRefGoogle Scholar
  10. 10.
    Kilby, N. J., Snaith, M. R., and Murray, J. A. H. (1993) Site-specific recombinase: tools for genome engineering.Trends Genet. 9, 413–421.PubMedCrossRefGoogle Scholar
  11. 11.
    Plasterk, R. H. A., Ilmer, T. A. M., and Van De Putte, P. (1983) Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions.Virology 127, 24–36.PubMedCrossRefGoogle Scholar
  12. 12.
    Tijhaar, E. J., Zheng-Xin, Y., Karlas, J. A., Meyer, T. F., Stukart, M. J., Osterhaus, A. D. M. E., and Mooi, F. R. (1994) Construction and evaluation of an expression vector allowing the stable expression of foreign antigens in aSalmonella typhimurium vaccine strain.Vaccine 12, 1004–1011.PubMedCrossRefGoogle Scholar
  13. 13.
    d'Oliveira, C., Tijhaar, E. J., Shiels, B. R., Van der Weide, M., and Jongejan, F. (1996) Expression of genes encoding two majorTheileria annulata merozoite surface antigens inEscherichia coli and aSalmonella typhimurium aroA vaccine strain.Gene 172, 33–39.CrossRefGoogle Scholar
  14. 14.
    Huber, H. E., Iida, S., and Bickle, T. A. (1985) Expression of the bacteriophage P1cin recombinase gene from its own and heterologous promoters.Gene 34, 63–72.PubMedCrossRefGoogle Scholar
  15. 15.
    Dale, E. C. and Ow, D. W. (1990) Intra-and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase.Gene 91, 79–85.PubMedCrossRefGoogle Scholar
  16. 16.
    Casabadan, M. J. (1980) Analysis of gene control signals by DNA fusion inEscherichia coli.J. Mol. Biol. 138, 179–207.CrossRefGoogle Scholar
  17. 17.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  18. 18.
    Pribnow, D. (1975) Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter.Proc. Natl. Acad. Sci. USA 72, 784–788.PubMedCrossRefGoogle Scholar
  19. 19.
    Lisser, S. and Margalit, H. (1993) Compilation ofE. coli mRNA promoter sequences.Nucleic Acids Res. 21, 1507–1516.PubMedCrossRefGoogle Scholar
  20. 20.
    Simons, R. W., Houman, F., and Kleckner, N. (1987) Improved single and multicopylac-based cloning vectors for protein and operon fusions.Gene 53, 85–96.PubMedCrossRefGoogle Scholar
  21. 21.
    Greener, A., Filutowicz, M. S., McEachern, M. J., and Helinski, D. R. (1990) N-terminal truncated forms of the bifuncional π initiation protein express negative activity on plasmid R6K replication.Mol. Gen. Genet. 224, 24–32.PubMedCrossRefGoogle Scholar
  22. 22.
    Durland, R. H., Toukdarian, A., Fang, F., and Helinski, D. R. (1990) Mutations in thetrfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers.J. Bacteriol. 172, 3859–3867.PubMedGoogle Scholar
  23. 23.
    Andrews, B. J., Proteau, G. U., Beatty, L. G., and Sadowski, P. D. (1985) The FLP recombinase of the 2μ circle DNA of yeast: interaction with its target sequences.Cell 40, 795–803.PubMedCrossRefGoogle Scholar
  24. 24.
    Miller, J. H. (1972)Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 352–355.Google Scholar
  25. 25.
    Wu, F., Goldberg, I., and Filutowicz, M. (1992) Roles of a 106-bp origin enhancer andEscherichia coli DnaA protein in replication of plasmid R6K.Nucleic Acids Res. 20, 811–817.PubMedCrossRefGoogle Scholar
  26. 26.
    Chang, A. C. Y. and Cohen, S. N. (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from P15A cryptic miniplasmid.J. Bacteriol. 134, 1141–1156.PubMedGoogle Scholar
  27. 27.
    Kim, J., Zwieb, C., Wu, C., and Adhya, S. (1989) Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector.Gene 85, 15–23.PubMedCrossRefGoogle Scholar
  28. 28.
    Senecoff, J. F., Bruckner, R. C., and Cox, M. M. (1985) The FLP recombinase of the yeast 2-μm plasmid: characterization of its recombination site.Proc. Natl. Acad. Sci. USA 82, 7270–7274.PubMedCrossRefGoogle Scholar
  29. 29.
    Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H., and Falkow, S. (1977) Construction and characterization of new cloning vehicles, II: a multipurpose cloning system.Gene 2, 95–113.PubMedCrossRefGoogle Scholar
  30. 30.
    Sutcliffe, J. F. (1979) Complete nucleotide sequence of theEscherichia coli plasmid pBR322.Cold Spring Harbor Symp. Quant. Biol. 43, 77–90.PubMedGoogle Scholar

Copyright information

© Humana Press Inc 1998

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of GdańskGdańskPoland
  2. 2.McArdle Laboratory for Cancer ResearchUniversity of Wisconsin Medical SchoolMadison

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