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Folia Microbiologica

, Volume 39, Issue 3, pp 171–175 | Cite as

Construction of recombinant K88 DNA with Ptac promoter

  • E. Holoda
  • I. Mikula
Papers

Abstract

The gene encoding K88ab was localized on the 11.6 kbHindIII-HindIII fragment of 74 kb plasmid DNA ofE. coli 7301. The smallest recombinant DNA producing the K88ab antigen was obtained by excision of the 5.15 kbEcoRI-EcoRI fragment from recombinant DNA composed of the 11.6 kb K88ab fragment in the pBR322 vectro. The size of the smallest fragment was 6.5 kb. Expression of the K88ab antigen was controlled by the P1 promoter of the pBR322 vector. Substitution of promoter Ptac for promoter P1 made it possible to achieve expression of the K88ab antigen byE. coli MT. Substitution of promoter PL for promoter P1 failed to achieve expression of the K88 ab antigen in the recipient strains used.

Keywords

Transformation Product Klenow Fragment Chymosin Thermal Induction pBR322 Vector 
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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References

  1. Ausubel F.M., Brent R., Kingston R.E., Moore D., Seidman J.G., Smith J.A., Struhl K.:Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Interscience, New York 1989.Google Scholar
  2. Birnboim H.C., Doly I.: A rapid alkaline extraction procedure for screening recombinant plasmid.Nucl. Acids Res. 7, 1513–1520 (1979).PubMedCrossRefGoogle Scholar
  3. Doskočil J.: Recombination of DNA molecules and expression. (In Czech)Biol. listy 43, 93–107 (1978).Google Scholar
  4. Dougan G., Dowd G., Kehoe M.: Organization of K88ac-encoded polypeptides in theE. coli envelope: Use of minicells and outer membrane protein mutants for studying assembly of pili.J. Bacteriol. 153, 364–370 (1983).PubMedGoogle Scholar
  5. Guinnée P.A.M., Jansen H.W.: Behavior ofE. coli K antigens K88ab, K88ac and K88ad in immunoelectrophoresis double diffusion and hemagglutination.Infect. Immun. 23, 700–705 (1979).Google Scholar
  6. Gyles C.L., Maas W.K.: Recombinant DNA technology and enterotoxigenicE. coli vaccines.Progr. Vet. Microbiol. Immunol. 3, 139–158 (1987).Google Scholar
  7. Holoda E., Mikula I.: Characterization of plasmid profile in attenuated salmonella strains.Folia Veterinaria 37 (1-2), 27–31 (1993).Google Scholar
  8. Kehoe M., Sellwood R., Shipley P., Dougan G.: Genetic analysis of K88-mediated adhesion of enterotoxigenicE. coli.Nature 291, 1222–1226 (1981).CrossRefGoogle Scholar
  9. Jacobs A.A.C., Roosendaal B., Van Breemen J.F.L., De Graaf F.K.: Role of phenylalanine 150 in the receptor-binding domain of the K88 fibrillar subunit.J. Bacteriol. 169, 4907–4911 (1987).PubMedGoogle Scholar
  10. Maniatis T., Fritsch E.F., Sambrook J.:Molecular Cloning-A Laboratory Manual, pp. 784–787. Cold Spring Harbor, New York 1982.Google Scholar
  11. Mooi F.R., De Graaf F.K.: Isolation and characterization of K88 antigens.FEMS Microbiol. Lett. 45, 17–20 (1980).Google Scholar
  12. Mooi F.R., Claassen I., Bakker D., Kuipers H., De Graaf F.K.: Regulation and structure of anE. coli gene coding for an outer membrane protein involved in export K88ab fimbrial subunits.Nucl. Acids Res. 14, 2443–2457 (1986).PubMedCrossRefGoogle Scholar
  13. Ørskov I., Ørskov F.: Episome-carried surface antigen K88 ofE. coli.J. Bacteriol. 91, 69–75 (1966).PubMedGoogle Scholar
  14. Schein C.H., Noteborn M.H.M.: Fermentation of soluble recombinant proteins inE. coli is favored by lower growth temperature.Biotechnology 6, 291–294 (1988).CrossRefGoogle Scholar
  15. Scott J.R.: Regulation of plasmid replication.Microbiol. Rev. 48, 1–23 (1984).PubMedGoogle Scholar
  16. Sedláček J., Fábry M., Kašpar P., Zadražil S., Kaprálek F.: Plasmid construct for overproduction of chymosin inE. coli.Biotechnol. Bioindustry 6, 17–19 (1986).Google Scholar
  17. Stüber D., Bujard H.: Organization of transcriptional signals in plasmids pBR322 and pACYC184.Proc. Nat. Acad. Sci. USA 78, 167–171 (1981).PubMedCrossRefGoogle Scholar
  18. Vanier P.: The issue of new generation vaccines hierarchy of the risk.Develop. Biol. Standard 79, 99–104 (1992).Google Scholar
  19. Wolfes H., Alves J., Fliess A., Geiger R., Pingoud A.: Site directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential for endonucleolytic activity ofEco RI.Nucl. Acids Res. 14, 9063–9080 (1986).PubMedCrossRefGoogle Scholar

Copyright information

© Folia Microbiologica 1994

Authors and Affiliations

  • E. Holoda
    • 1
  • I. Mikula
    • 1
  1. 1.Department of Microbiology and ImmunologyUniversity of Veterinary MedicineKošiceSlovak Republic

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