pBT bait plasmid双杂交系统质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pBT bait plasmid双杂交系统质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
The pBT bait plasmid and the pTRG target plasmid are designed to allow
detection of protein-protein interactions when used to cotransform a host
strain containing the appropriate reporter gene cassette. The 3.2 kb pBT bait
plasmid (Figure 2) carries a low-copy p15A replication origin and confers
chloramphenicol resistance. The plasmid encodes the full-length bacterial
phage λ cI protein under the control of the IPTG-inducible lac-UV5
promoter. The multiple cloning site* contains several restriction sites to
facilitate fusion gene construction. From a 5 ml LB-chloramphenicol culture
(34 μg/ml chloramphenicol), yields of approximately 200 ng are obtained.
The 4.4 kb target plasmid, pTRG (Figure 3), carries the low-copy ColE1
replication origin and confers tetracycline resistance. The plasmid directs
transcription of the amino-terminal domain of RNA polymerase α subunit
through a multiple cloning site at the 3´ end of the α subunit gene and is
under the control of the IPTG-inducible, tandem promoter lpp/lac-UV5. The
expression level from the lpp/lac-UV5 promoter operator is higher than the
levels of lac-UV5-driven expression. The arrangement of EcoR I and Xho I
restriction sites in the pTRG MCS makes the plasmid compatible with
inserts produced using the Stratagene cDNA Synthesis Kit (Catalog
#200401). From a 5 ml LB-tetracycline culture (12.5 μg/ml tetracycline),
yields of approximately 1 μg are obtained.
Both bait and target plasmids contain the Not I restriction site in the MCS
which encodes a short alanine linker to facilitate the orientation and folding
of the fused bait and target proteins.
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