Question

The BL21(DE3) expression cells carry a λDE3 integration containing the T7-RNA polymerase gene.

A) Why is the T7-RNA polymerase protein so specific with regard to the target genes it transcribes?

B) Is this enzyme predicted to transcribe any E. coli genes? Why or why not?

Answer 1

a) In the pET vectors, the target gene is under the control of a T7- promoter, so only a T7 RA polymerase can lead to the transcription & eventually expression of the target gene present in the vector. So, the T& RNA polymerase gene is integrated in the genome of the host BL21(DE3) cells so that T7 RNA polymerase is expressed which will eventually lead to the expression of the target gene in the vector plasmid.

b) - The T7 RNA polymerase is under the control of a lac operator-promoter in the genome, so only due to induction of TPTG, the T7 RNA polymerase will be expressed. Hence by controlling the expression of the T7 RNA polymersae, we can effectively control the exression of the target gene in the plasmid.
Also the target gene is only under the control of the T7 promoter (a viral promoter, obtained from T7 phage)- no other gene in the bacterial cells in under its control. So T7 RNA polymerase will not transcribe any other E.coli genes.