Bacillus megaterium pPT7 cloning vector巨大芽孢杆菌表达载体质粒 BioVector NTCC质粒载体菌种细胞基因保藏中心

Bacillus megaterium pPT7 cloning vector巨大芽孢杆菌表达载体质粒

The T7 RNA polymerase (T7 RNAP) expression system originates from the
bacteriophage DNA-dependent RNA polymerase. In 1985, the first described T7 RNAP
expression system was developed for Escherichia coli (Tabor and Richardson, 1985).
Advantages of this system are the stringent selectivity and the high transcriptional activity
so that it is possible to lead to a saturation of the protein-synthesizing machinery in E.
coli. Consequently, 50% or more of the total cellular protein can consist of the desired
protein (Studier and Moffatt, 1986).
The T7 RNAP expression system for B. megaterium combines the features of this system
in E. coli with the above mentioned regulation by the xylose operon. This 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 for ampicillin and chloramphenicol resistance 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 comprises two
resistances against ampicillin (in E. coli) and tetracycline (in B. megaterium).
Next to the pPT7 vector, we offer an expression vector that leads to secretion of
expressed proteins into the surrounding medium. pPT7-SPLipA provides the n-terminal
signal peptide LipA that uses the Sec-pathway for protein secretion.
Furthermore, a great obstacle in protein production and engineering, the cloning of genes
coding for proteins that are toxic to the cloning host E. coli, can be overcome by means
of the T7 expression system due to the absence of T7 RNAP in the cloning host.
For your convenience MoBiTec offers 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.

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