• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Mar 15, 1997; 25(6): 1203–1210.
PMCID: PMC146584

Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements.

Abstract

Based on parameters governing promoter activity and using regulatory elements of the lac, ara and tet operon transcription control sequences were composed which permit the regulation in Escherichia coli of several gene activities independently and quantitatively. The novel promoter PLtetO-1 allows the regulation of gene expression over an up to 5000-fold range with anhydrotetracycline (aTc) whereas with IPTG and arabinose the activity of Plac/ara-1 may be controlled 1800-fold. Escherichia coli host strains which produce defined amounts of the regulatory proteins, Lac and Tet repressor as well as AraC from chromosomally located expression units provide highly reproducible in vivo conditions. Controlling the expression of the genes encoding luciferase, the low abundance E.coli protein DnaJ and restriction endonuclease Cfr9I not only demonstrates that high levels of expression can be achieved but also suggests that under conditions of optimal repression only around one mRNA every 3rd generation is produced. This potential of quantitative control will open up new approaches in the study of gene function in vivo, in particular with low abundance regulatory gene products. The system will also provide new opportunities for the controlled expression of heterologous genes.

Full Text

The Full Text of this article is available as a PDF (230K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bukau B. Regulation of the Escherichia coli heat-shock response. Mol Microbiol. 1993 Aug;9(4):671–680. [PubMed]
  • Erickson HP. FtsZ, a prokaryotic homolog of tubulin? Cell. 1995 Feb 10;80(3):367–370. [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. [PMC free article] [PubMed]
  • Elvin CM, Thompson PR, Argall ME, Hendry P, Stamford NP, Lilley PE, Dixon NE. Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene. 1990 Mar 1;87(1):123–126. [PubMed]
  • Giordano TJ, Deuschle U, Bujard H, McAllister WT. Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system. Gene. 1989 Dec 14;84(2):209–219. [PubMed]
  • Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. [PMC free article] [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Brosius J, Erfle M, Storella J. Spacing of the -10 and -35 regions in the tac promoter. Effect on its in vivo activity. J Biol Chem. 1985 Mar 25;260(6):3539–3541. [PubMed]
  • Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121–4130. [PMC free article] [PubMed]
  • Skerra A. Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene. 1994 Dec 30;151(1-2):131–135. [PubMed]
  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Lanzer M, Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973–8977. [PMC free article] [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Bonin AL, Gossen M, Bujard H. Photinus pyralis luciferase: vectors that contain a modified luc coding sequence allowing convenient transfer into other systems. Gene. 1994 Apr 8;141(1):75–77. [PubMed]
  • Cohen SN, Chang AC. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol. 1977 Nov;132(2):734–737. [PMC free article] [PubMed]
  • Diederich L, Rasmussen LJ, Messer W. New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome. Plasmid. 1992 Jul;28(1):14–24. [PubMed]
  • Manen D, Xia G, Caro L. A locus involved in the regulation of replication in plasmid pSC101. Mol Microbiol. 1994 Mar;11(5):875–884. [PubMed]
  • Kammerer W, Deuschle U, Gentz R, Bujard H. Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process. EMBO J. 1986 Nov;5(11):2995–3000. [PMC free article] [PubMed]
  • Philipp A, Schneider A, Väsrik I, Finke K, Xiong Y, Beach D, Alitalo K, Eilers M. Repression of cyclin D1: a novel function of MYC. Mol Cell Biol. 1994 Jun;14(6):4032–4043. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Lubys A, Menkevicius S, Timinskas A, Butkus V, Janulaitis A. Cloning and analysis of translational control for genes encoding the Cfr9I restriction-modification system. Gene. 1994 Apr 8;141(1):85–89. [PubMed]
  • de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Deuschle U, Kammerer W, Gentz R, Bujard H. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986 Nov;5(11):2987–2994. [PMC free article] [PubMed]
  • Hillen W, Berens C. Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol. 1994;48:345–369. [PubMed]
  • Brunner M, Bujard H. Promoter recognition and promoter strength in the Escherichia coli system. EMBO J. 1987 Oct;6(10):3139–3144. [PMC free article] [PubMed]
  • Oehler S, Amouyal M, Kolkhof P, von Wilcken-Bergmann B, Müller-Hill B. Quality and position of the three lac operators of E. coli define efficiency of repression. EMBO J. 1994 Jul 15;13(14):3348–3355. [PMC free article] [PubMed]
  • Oehler S, Eismann ER, Krämer H, Müller-Hill B. The three operators of the lac operon cooperate in repression. EMBO J. 1990 Apr;9(4):973–979. [PMC free article] [PubMed]
  • Shore D, Baldwin RL. Energetics of DNA twisting. I. Relation between twist and cyclization probability. J Mol Biol. 1983 Nov 15;170(4):957–981. [PubMed]
  • Gentz R, Langner A, Chang AC, Cohen SN, Bujard H. Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4936–4940. [PMC free article] [PubMed]
  • Müller-Hill B, Crapo L, Gilbert W. Mutants that make more lac repressor. Proc Natl Acad Sci U S A. 1968 Apr;59(4):1259–1264. [PMC free article] [PubMed]
  • Zhang X, Bremer H. Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia coli. J Mol Biol. 1996 May 31;259(1):27–40. [PubMed]
  • Sancar A, Hack AM, Rupp WD. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. [PMC free article] [PubMed]
  • Degenkolb J, Takahashi M, Ellestad GA, Hillen W. Structural requirements of tetracycline-Tet repressor interaction: determination of equilibrium binding constants for tetracycline analogs with the Tet repressor. Antimicrob Agents Chemother. 1991 Aug;35(8):1591–1595. [PMC free article] [PubMed]
  • Stoltzfus L, Wilcox G. Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression. J Bacteriol. 1989 Feb;171(2):1178–1184. [PMC free article] [PubMed]
  • Oehmichen R, Klock G, Altschmied L, Hillen W. Construction of an E. coli strain overproducing the Tn10-encoded TET repressor and its use for large scale purification. EMBO J. 1984 Mar;3(3):539–543. [PMC free article] [PubMed]
  • Wissmann A, Meier I, Wray LV, Jr, Geissendörfer M, Hillen W. Tn10 tet operator mutations affecting Tet repressor recognition. Nucleic Acids Res. 1986 May 27;14(10):4253–4266. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...