5 replies to this topic

[WHR]

Hello everyone,
What is the purpose of T7 and sp6 promoter in a TA cloning vector?
Are they sufficient for inserted gene expression in BL21(DE3) E coli?

[hanming86]

WHR, on May 1 2009, 01:57 AM, said:

Hello everyone,
What is the purpose of T7 and sp6 promoter in a TA cloning vector?
Are they sufficient for inserted gene expression in BL21(DE3) E coli?

There're primer for those sites to aid in sequencing ur gene of interest

and yes the promoter seq will do some great expression in BL21 assuming that your sequence is in frame after confirming it with sequencing

[WHR]

hanming86, on May 1 2009, 06:37 AM, said:

There're primer for those sites to aid in sequencing ur gene of interest

and yes the promoter seq will do some great expression in BL21 assuming that your sequence is in frame after confirming it with sequencing

Thank you hanming86.

[bob1]

They are actually RNA polymerase binding sites designed put there to allow expression of the cloned gene. Not just there for primer binding.

[HomeBrew]

However, if you rely on them, you'll need to have a host strain that expresses either the T7 or the Sp6 RNA polymerase, depending on which promoter you want to use. Since the lambda DE3 lysogen carries the T7 RNA polymerase, the BL21(DE3) strain is useful for expression of a gene cloned in this vector, provided it's in the correct orientation. There is no frame for transcription, only for translation.

[Dr Teeth]

As Bob1 said, these are polymerase binding sites typically used for in vitro transcription/translation reactions where the Sp6 or T7 polymerase is added to the mix in a rabbit reticulocyte or wheat germ background.