For example, in the pDEST17, the ccdB gene is under expression of the T7 promoter. I know this promoter requires the T7 RNA polymerase for expression. However, there is no T7 RNA polymerase gene on the pDEST, so in theory, you would either need an E. coli strain that contains the T7 RNA polymerase gene or you would need to infect your cells with a T7 bacteriophage that expresses the T7 RNA polymerase. But how come I can't find anything on this matter in the protocol? Could the ccdB gene also be active without the T7 promoter?
And is it actually needed to first transform into the DH5alpha strain and then do the expression in the BL21 strain? Why not immediately transform into BL21?
The CcdB coding sequence in all the Gateway vectors actually has its own promoter just in front of it, this is the reason it can expressed in any bacterium strain. This promoter is very week and not good enough to drive the target protein expression, for that reason the pDEST17 is engineered a T7 promoter for target protein expression. In recombination, you do not always get high efficiency and you need some E. coli cells with high competency to select the recombinants. In comparison with the cloning competent cells such as DH5 alpha, expression strains such as BL21 usually are less competent. Another reason is the cloning strains are favorite for plasmid replication of good yield and high quality, you need this for DNA manupulation e.g. DNA sequenceing, while the expression strains are favourite for transcription and protein expression and usually produce less plasmid DNA.
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