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. 1990 Jan;34(1):111–116. doi: 10.1128/aac.34.1.111

Sensitive biological detection method for tetracyclines using a tetA-lacZ fusion system.

I Chopra 1, K Hacker 1, Z Misulovin 1, D M Rothstein 1
PMCID: PMC171530  PMID: 2158271

Abstract

A sensitive microbiological detection system for tetracyclines, utilizing an Escherichia coli strain containing a cloned tetA-lacZ gene fusion, is described. Expression of beta-galactosidase by the fusion plasmid pUB3610 remained subject to regulatory control by the TetR repressor protein, with the presence of tetracyclines in the growth medium leading to a 12-fold induction of beta-galactosidase synthesis. Because synthesis of beta-galactosidase was influenced to a small extent by the carbon source and the addition of cyclic AMP to the medium, cells were grown in the presence of cyclic AMP to enhance the sensitivity of the assay. All commonly marketed tetracyclines and some derivatives at concentrations as low as 0.1 ng/ml could be detected in the growth medium. A plate assay utilizing the fusion plasmid that detects 1 ng of tetracycline has also been developed.

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Selected References

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  1. Bernardi A., Bernardi F. Complete sequence of pSC101. Nucleic Acids Res. 1984 Dec 21;12(24):9415–9426. doi: 10.1093/nar/12.24.9415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bertrand K. P., Postle K., Wray L. V., Jr, Reznikoff W. S. Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant. J Bacteriol. 1984 Jun;158(3):910–919. doi: 10.1128/jb.158.3.910-919.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  5. Chatten L. G., Moskalyk R. E., Locock R. A., Huang K. S. Dc Polarographic assay of tetracyclines. J Pharm Sci. 1976 Sep;65(9):1315–1319. doi: 10.1002/jps.2600650913. [DOI] [PubMed] [Google Scholar]
  6. Chopra I., Howe T. G., Linton A. H., Linton K. B., Richmond M. H., Speller D. C. The tetracyclines: prospects at the beginning of the 1980s. J Antimicrob Chemother. 1981 Jul;8(1):5–21. doi: 10.1093/jac/8.1.5. [DOI] [PubMed] [Google Scholar]
  7. Chopra I., Johnson S. C., Bennett P. M. Inhibition of Providencia stuartii cell envelope enzymes by chlorhexidine. J Antimicrob Chemother. 1987 Jun;19(6):743–751. doi: 10.1093/jac/19.6.743. [DOI] [PubMed] [Google Scholar]
  8. Dixon R. A., Chopra I. Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide. Antimicrob Agents Chemother. 1986 May;29(5):781–788. doi: 10.1128/aac.29.5.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eccles S. J., Chopra I. Biochemical and genetic characterization of the tet determinant of Bacillus plasmid pAB124. J Bacteriol. 1984 Apr;158(1):134–140. doi: 10.1128/jb.158.1.134-140.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elespuru R. K., White R. J. Biochemical prophage induction assay: a rapid test for antitumor agents that interact with DNA. Cancer Res. 1983 Jun;43(6):2819–2830. [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Ivanov I. G., Bachvarov D. R. Determination of plasmid copy number by the "boiling" method. Anal Biochem. 1987 Aug 15;165(1):137–141. doi: 10.1016/0003-2697(87)90211-9. [DOI] [PubMed] [Google Scholar]
  13. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Katz J. M., Katz S. E. Rapid assay for tetracycline in premixes and mixed feeds. J Assoc Off Anal Chem. 1984 May-Jun;67(3):576–579. [PubMed] [Google Scholar]
  15. Klock G., Unger B., Gatz C., Hillen W., Altenbuchner J., Schmid K., Schmitt R. Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants. J Bacteriol. 1985 Jan;161(1):326–332. doi: 10.1128/jb.161.1.326-332.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pastan I., Perlman R. L. The role of the lac promotor locus in the regulation of beta-galactosidase synthesis by cyclic 3',5'-adenosine monophosphate. Proc Natl Acad Sci U S A. 1968 Dec;61(4):1336–1342. doi: 10.1073/pnas.61.4.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Peden K. W. Revised sequence of the tetracycline-resistance gene of pBR322. Gene. 1983 May-Jun;22(2-3):277–280. doi: 10.1016/0378-1119(83)90112-9. [DOI] [PubMed] [Google Scholar]
  18. Samra Z., Krausz-Steinmetz J., Sompolinsky D. Transport of tetracyclines through the bacterial cell membrane assayed by fluorescence: a study with susceptible and resistant strains of Staphylococcus aureus and Escherichia coli. Microbios. 1978;21(83):7–21. [PubMed] [Google Scholar]
  19. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  20. Sultan S. M. Spectrophotometric determination of tetracycline with sodium molybdate. Analyst. 1986 Jan;111(1):97–99. doi: 10.1039/an9861100097. [DOI] [PubMed] [Google Scholar]
  21. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  22. Watson N. A new revision of the sequence of plasmid pBR322. Gene. 1988 Oct 30;70(2):399–403. doi: 10.1016/0378-1119(88)90212-0. [DOI] [PubMed] [Google Scholar]
  23. de Crombrugghe B., Busby S., Buc H. Cyclic AMP receptor protein: role in transcription activation. Science. 1984 May 25;224(4651):831–838. doi: 10.1126/science.6372090. [DOI] [PubMed] [Google Scholar]
  24. van den Bogert C., Kroon A. M. Fluorometric determination of tetracyclines in small blood and tissue samples. J Pharm Sci. 1981 Feb;70(2):186–189. doi: 10.1002/jps.2600700218. [DOI] [PubMed] [Google Scholar]

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