Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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T7 RNA Polymerase High Yield Gene Expression System for Bacillus megaterium

High Yield Gene Expression with our T7 RNA Polymerase Expression System

 Vector map of pPT7

Vector map of pPT7

Features

  • Tightly regulated and efficiently inducible xylA operon/T7 RNA polymerase
  • Strong T7 RNAP promoter with unique sequence
  • Stable, high yield protein production up to 8-fold increased in comparison to common xylose inducible expression
  • Easy transformation by use of pretransformed B. megaterium protoplasts
  • Control vector with GFP-sequence included in the kit
  • Secretion vector is also available

Description

  • This system combines the benefits of the tightly regulated and strong T7 RNA polymerase expression system and alkaline protease free Bacillus megaterium. Our system is based on two parallel-replicating plasmids: pT7-RNAP and pPT7 (Gamer et al. 2009). In addition to the t7 rnap gene under control of the strong xylA promoter pT7-RNAP contains the genes of ampicillin and chloramphenicol for easy selection in E. coli (AmpR) and B. megaterium (CmR). pPT7 is responsible for the T7 RNAP-dependent expression of the target gene. Downstream of the T7 promoter it comprises a multiple cloning site with ten unique restriction enzyme cleaving sites. Additionally, the plasmid carries two resistances to ampicillin (for E .coli) and tetracycline (for B. megaterium).
 Vector map of pT7-RNAP

Vector map of pT7-RNAP

Comparison of GFP amounts produced by variant expression systems. GFP produced by B. megaterium carrying the common xylose-regulated plasmids pRBBm34 (square), B. megaterium transformed with the T7 expression system plasmids (circle) and B. megaterium with the T7 expression system plasmids treated with rifampicin. (Gamer et al., 2009)

Secretion vector pPT7-SP LipA

The vector pPT7-SPLipA provides the Lipase A signal sequence that leads to secretion of your protein of interest into the medium.

Pretransformed B. megaterium protoplasts

For your convenience we offer B. megaterium protoplasts pretransformed with pT7-RNAP, so that you just have to insert your gene of interest into pPT7 and transform the pretransformed protoplasts with this plasmid. For control purposes the GFP-expressing vector pPT7-GFP is included in the kit.

Video

This video shows a growing micro-colony of cells of Bacillus megaterium MS941 expressing GFP observed in a Zeiss Axiovert 200 M microscope. The cells are induced shortly before the video starts. A brightfield image and a fluorescence image are recorded every 5 minutes over a timespan of 5 1/2 hours. For this video the brightfield images are overlayed by the green colored fluorescence images. Recorded by Stefan Leupold, Institut für Mikrobiologie, Technische Universität Braunschweig.

ORDER INFORMATION

  • For shipping and storage information please click on Order#.
Order# Description Amount Price Data Sheet
BMEGT702 Bacillus megaterium protoplasts, strain MS941, pretransformed with pT7-RNAP 5x500 µl 684,00 PDF
BMEGT701 Bacillus megaterium high yield T7 gene expression kit, includes pretransformed protoplasts BMEGT702 (5x500µl), pPT7 cloning vector and pPT7-GFP control vector (vectors lyophilized, 10 µg each) Kit 1143,00 PDF
BMEGT710 Bacillus megaterium pPT7 cloning vector, lyophilized 10 µg 619,00 PDF
BMEGT711 Bacillus megaterium pPT7-SPlipA secretion vector 10 µg 678,00 PDF
BMEG50 Bacillus megaterium protoplasts, strain MS941 5x500 µl 464,00 PDF
  • All prices are in EURO excl. VAT and shipping. For further pricing and order information please ask your local distributor.

  • Vectors are E. coli / B. megaterium shuttle vectors
  • Please note that protoplasts of Bacillus megaterium have a limited shelf life of some months only, depending on the date of production. Therefore purchase and use of protoplasts must be thoroughly planned.

Download

T7 RNAP Expression System Handbook (PDF)
Commentary – A short story about a big magic bug (Link)

Vector maps and Sequences

Vector map of pPT7 (pic)
Vector map of pPT7-GFP (pic)
Vector map of pPT7-SPLipA (pic)
Vector map of pT7-RNAP (pic)

Sequence of pPT7 (txt)
Sequence of pPT7-GFP (txt)
Sequence of pPT7-SPLipA (txt)
Sequence of pT7-RNAP (txt)

Literature

  1. Gamer, M., Fröde, D., Biedendieck, R., Stammen, S., und Jahn, D. (2009). A T7 RNA polymerase-dependent gene expression system for Bacillus megaterium. Appl. Microbiol. Biotechnol 82, 1195-1203.
  2. Biedendieck, R., Beine, R., Gamer, M., Jordan, E., Buchholz, K., Seibel, J., Dijkhuizen, L., Malten, M., und Jahn, D. (2007). Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium. Appl. Microbiol. Biotechnol 74, 1062-1073.
  3. Biedendieck, R., Gamer, M., Jaensch, L., Meyer, S., Rohde, M., Deckwer, W., und Jahn, D. (2007). A sucrose-inducible promoter system for the intra- and extracellular protein production in Bacillus megaterium. J. Biotechnol 132, 426-430.
  4. Biedendieck, R., Yang, Y., Deckwer, W., Malten, M., und Jahn, D. (2007). Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium. Biotechnol. Bioeng 96, 525-537.
  5. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  6. Puyet, A., Sandoval, H., López, P., Aguilar, A., Martin, J., und Espinosa, M. (1987). A simple medium for rapid regeneration of Bacillus subtilis protoplasts transformed with plasmid DNA. FEMS Microbiology Letters 40, 1-5.
  7. Rygus, T., und Hillen, W. (1991). Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl. Microbiol. Biotechnol 35, 594-599.
  8. Rygus, T., Scheler, A., Allmansberger, R., und Hillen, W. (1991). Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Arch. Microbiol 155, 535-542.
  9. Studier, F. W., und 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.
  10. Tabor, S., und 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.