Expression Using the T7 RNA Polymerase/Promoter System

Stanley Tabor1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.2
DOI:  10.1002/0471142727.mb1602s11
Online Posting Date:  May, 2001
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Abstract

This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (pT7). In the , two plasmids are maintained within the same E. coli cell. One (the expression vector) contains pT7 upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heatā€inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of pT7. If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.

     
 
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Table of Contents

  • Basic Protocol 1: Expression Using the Two‐Plasmid System
  • Alternate Protocol 1: Selective Labeling of Plasmid‐Encoded Proteins
  • Alternate Protocol 2: Expression by Infection with M13 Phage mGP1‐2
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Expression Using the Two‐Plasmid System

  Materials
  • pT7‐5, pT7‐6, or pT7‐7 vectors (available from author)
  • E. coli JM105, DH1, or equivalent (Table 97.80.4711)
  • LB plates and medium containing 60 µg/ml ampicillin (unit 1.1)
  • E. coli K38 or equivalent (Table 97.80.4711)
  • pGP1‐2 (available from author)
  • LB plates and medium containing 60 µg/ml kanamycin (unit 1.1)
  • LB plates and medium containing 60 µg/ml ampicillin plus 60 µg/ml kanamycin (unit 1.1)
  • recipeCracking buffer
  • Sorvall SS‐34 or GS‐3 rotor or equivalent
  • Additional reagents and equipment for subcloning DNA fragments (units 1.4 & 3.16), transformation of competent E. coli cells (unit 1.8), minipreps of plasmid DNA (unit 1.6), restriction mapping (units 3.1 3.3), and SDS‐PAGE (unit 10.210.2).

Alternate Protocol 1: Selective Labeling of Plasmid‐Encoded Proteins

  Additional Materials
  • M9 medium (unit 1.1) without and with 5% (v/v) of recipe18 amino acid mixture
  • 20 mg/ml rifampicin in methanol (e.g., Sigma #R‐3501; store in dark at 4°C for 2 weeks; Table 97.80.4711)
  • 10 mCi/ml [35S]methionine (>800 Ci/mmol) diluted 1:10 in M9 medium
  • Fluorographic enhancing agent (e.g., Enlightning from Du Pont NEN or Amplify from Amersham)

Alternate Protocol 2: Expression by Infection with M13 Phage mGP1‐2

  Additional Materials
  • M13 phage mGP1‐2 (available from author)
  • PEG solution (unit 1.7)
  • 100 mM IPTG (Table 97.80.4711)
  • Additional reagents and equipment for preparing M13 phage (unit 1.15) and titering phage (unit 1.11).
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Figures

Videos

Literature Cited

Literature Cited
   Chen, W., Tabor, S., and Struhl, K. 1987. Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. Cell 50:1047‐1055.
   Dougan, G. and Sherratt, D. 1977. The transposon Tn1 as a probe for studying ColE1 structure and function. Mol. Gen. Genet. 151:151‐160.
   Dunn, J.J., Krippl, B., Bernstein, K.E., Westphal, H., and Studier, F.W. 1988. Targeting bacteriophage T7 RNA polymerase to the mammalian cell nucleus. Gene 68:259‐266.
   Fuerst, T.R., Niles, E.G., Studier, F.W., and Moss, B. 1986. Eukaryotic transient‐expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 83:8122‐8126.
   Kirel, P.‐H., Schmitter, J.‐M., Dessen, P., Fayat, G., and Blanquet, S. 1989. Extent of N‐terminal methionine excision from Escherichia coli proteins is governed by the side‐chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. U.S.A. 86:8247‐8251.
   Nakai, H. and Richardson, C.C. 1986. Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. J. Biol. Chem. 261:15208‐15216.
   Rosenberg, A.H., Lade, B.N., Chui, D., Lin, S., Dunn, J.J., and Studier, F.W. 1987. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125‐135.
   Russel, M. and Model, P. 1984. Replacement of the flp gene of Escherichia coli by an inactive gene cloned on a plasmid. J. Bacteriol. 159:1034‐1039.
   Sancar, A., Wharton, R.P., Seltzer, S., Kacinsky, B.M., Clark, N.D., and Rupp, W.D. 1981. Identification of the uvrA. gene product. J. Mol. Biol. 184:45‐62.
   Studier, F.W. and Moffatt, B.A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high‐level expression of cloned genes. J. Mol. Biol. 189:113‐130.
   Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. 1990. Use of T7 RNA polymerase to direct the expression of cloned genes. Methods Enzymol. 185. In press.
   Tabor, S. and Richardson, C.C. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U.S.A. 82:1074‐1078.
   Tabor, S. and Richardson, C.C. 1987. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 84:4767‐4771.
Key References
   Studier et al., 1990. See above.
  Gives extensive list of vectors and protocols for expression using T7 RNA polymerase.
   Tabor and Richardson, 1985. See above.
  Describes the use of the two‐plasmid system for expression of genes using T7 RNA polymerase.
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