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B. megaterium T7 RNA Polymerase High Yield Gene Expression

BMEGT701 - Bacillus megaterium high yield T7 gene expression kit

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The Bacillus megaterium high yield T7 gene expression system combines the features of the B. megaterium system in E. coli with the T7 RNA polymerase expression system regulation by the xylose operon. This dual plasmid system is based on two parallel-replicating plasmids: pT7-RNAP and pPT7pT7-RNAP contains the genes for ampicillin and chloramphenicol resistance for easy selection in E. coli (AmpR) and B. megaterium (CmR), in addition to the t7 rnap gene under control of the strong xylA promoter. 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, pPT7 comprises two resistances against ampicillin (in E. coli) and tetracycline (in B. megaterium).

B. megaterium has proven to be an excellent host for the expression of non-homologous DNA. Unlike other bacilli strains, none of the alkaline proteases are present and no endotoxins are found in the cell wall. B. megaterium can stably maintain several extra-chromosomal DNA elements in parallel. Protein yields are exceptionally high, even if inexpensive substrates are used. 

This kit includes strain MS941, pPT7 cloning vector, and pPT7-GFP control vector. A secretion vector, pPT7-SPLipA, is separately available. For added convenience, strain MS941 comes pretransformed with pT7-RNAP. This allows the user to immediately insert the gene of interest into pPT7 and transform the pretransformed protoplasts with this plasmid.

Please note this product requires a license for non-academic institutions. Please contact us for licensing information.


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.

Features

  • Strong T7 RNAP promoter with unique sequence
  • Tightly regulated and efficiently inducible xylA operon/T7 RNA polymerase
  • Suitable for cloning of toxic proteins in E. coli
  • Stable, high yield protein production up to 8-fold increase compared to common xylose inducible expression
  • Easy transformation by use of pretransformed B. megaterium protoplast
  • Ideal for both small- and large-scale protein production
  • B. megaterium is not pathogenic, no endotoxins found in cell wall
  • Extracellular protein production possible with pPT7-SPlipA (sold separately)

Contents

  • Pretransformed B. megaterium protoplast, strain MS941, 500µl x 5
  • B. megaterium pPT7 cloning vector, lyophilized, 10 µg 
  • B. megaterium pPT7-GFP control vector, lyophilized, 10 µg 


Literature