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. 1985 Apr;162(1):441–444. doi: 10.1128/jb.162.1.441-444.1985

Use of transcriptional repressors to stabilize plasmid copy number of transcriptional fusion vectors.

P F Lambert, W S Reznikoff
PMCID: PMC219010  PMID: 3920207

Abstract

Strong promoters cloned into transcriptional fusion vectors can adversely affect plasmid copy number. In this study, we investigated the use of transcriptional repressors, lacI and tetR, to stabilize the copy number of plasmids containing the lacUV5 and tetA promoters, respectively. Repression of these promoters was found to prevent plasmid copy number variation. Transcriptional strength of these promoters, when cloned into transcriptional fusion vectors, was determined by measuring the rate of synthesis after derepression with inducer. By using this approach, promoter strength can be accurately measured in vivo, without the need to compensate for copy number variation.

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Selected References

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  1. Adams C. W., Hatfield G. W. Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors. J Biol Chem. 1984 Jun 25;259(12):7399–7403. [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. Brosius J. Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators. Gene. 1984 Feb;27(2):161–172. doi: 10.1016/0378-1119(84)90137-9. [DOI] [PubMed] [Google Scholar]
  4. Hanau R., Koduri R. K., Ho N., Brenchley J. E. Nucleotide sequence of the control regions for the glnA and glnL genes of Salmonella typhimurium. J Bacteriol. 1983 Jul;155(1):82–89. doi: 10.1128/jb.155.1.82-89.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kenyon C. J., Brent R., Ptashne M., Walker G. C. Regulation of damage-inducible genes in Escherichia coli. J Mol Biol. 1982 Sep 25;160(3):445–457. doi: 10.1016/0022-2836(82)90307-2. [DOI] [PubMed] [Google Scholar]
  7. Miki T., Easton A. M., Rownd R. H. Cloning of replication, incompatibility, and stability functions of R plasmid NR1. J Bacteriol. 1980 Jan;141(1):87–99. doi: 10.1128/jb.141.1.87-99.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Munson L. M., Mandecki W., Caruthers M. H., Reznikoff W. S. Oligonucleotide mutagenesis of the lacPUV5 promoter. Nucleic Acids Res. 1984 May 11;12(9):4011–4017. doi: 10.1093/nar/12.9.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Reznikoff W. S., Michels C. A., Cooper T. G., Silverstone A. E., Magasanik B. Inhibition of lacZ gene translation initiation in trp-lac fusion strains. J Bacteriol. 1974 Mar;117(3):1231–1239. doi: 10.1128/jb.117.3.1231-1239.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Stueber D., Bujard H. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1982;1(11):1399–1404. doi: 10.1002/j.1460-2075.1982.tb01329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Tsao S. G., Brunk C. F., Pearlman R. E. Hybridization of nucleic acids directly in agarose gels. Anal Biochem. 1983 Jun;131(2):365–372. doi: 10.1016/0003-2697(83)90185-9. [DOI] [PubMed] [Google Scholar]
  12. Warren G. J., Twigg A. J., Sherratt D. J. ColE1 plasmid mobility and relaxation complex. Nature. 1978 Jul 20;274(5668):259–261. doi: 10.1038/274259a0. [DOI] [PubMed] [Google Scholar]
  13. Wray L. V., Jr, Reznikoff W. S. Identification of repressor binding sites controlling expression of tetracycline resistance encoded by Tn10. J Bacteriol. 1983 Dec;156(3):1188–1191. doi: 10.1128/jb.156.3.1188-1191.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yang H. L., Zubay G., Levy S. B. Synthesis of an R plasmid protein associated with tetracycline resistance is negatively regulated. Proc Natl Acad Sci U S A. 1976 May;73(5):1509–1512. doi: 10.1073/pnas.73.5.1509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Yu X. M., Reznikoff W. S. Deletion analysis of the CAP-cAMP binding site of the Escherichia coli lactose promoter. Nucleic Acids Res. 1984 Jul 11;12(13):5449–5464. doi: 10.1093/nar/12.13.5449. [DOI] [PMC free article] [PubMed] [Google Scholar]

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