pET expression vectors Flashcards
what is the T7 promoter?
not naturally found in E.Coli
only driven by T7 RNA polymerase - proteins expressed by T7P may comprise >50% of total cell protein
what are the features of T7P?
large number mRNA/S (high speed transcription)
high regulation of recombinant expression (switch on/off) - important as it can slow or prevent replication (some proteins are highly toxic and bind to DNA) - “cell factory technology”
what is the amp resistance gene for and why is kanamycin resistance best?
amp resistance for selection of cells that have uptaken plasmid (selectable marker)
Kan = better - contains origin of autonomous replication - high copy number of plasmid (large number of mRNA/S promoted by T7P RBS)
what is the IPTG inducer?
not metabolised by E.Coli - Inactivated by Lac repressor (gratuitous repressor) added at mid log phase
what is low level basal (leaky) expression?
in absence of IPTG there can be some expression of T7 RNA pol from the lacP on the E. coli BL21(DE3) genome
problematic if recombinant gene cloned into pET vector is highly toxic - may prevent cell growth to mid log phase
how may recombinant proteins be induced by bacteriophage CE6?
Use a non-BL21(DE3) E. coli host carrying a recombinant pET vector
T7 RNA pol can be introduced by infection with bacteriophage CE6 which carries the cloned T7 RNA pol gene
why carry lac repressor on pET vector?
high copy number - required to bind to operator sites to repress expression
