Why does the pET- expression vector contain a LacI gene additionally to the one in the genome? The pET plasmid is used for protein expression with T7 promotor in expression strains, such as _E.coli_ BL21(DE3) It contains a lacI gene which codes for the lac repressor protein, a protein of interest under the control of a T7 promoter for T7 RNA polymerase and a lac operator which can block transcription, directly behind the promotor. The lac operon is another control of transcription of the protein of interest. Furthermore, T7 polymerase is located in the genome as well under the control of the lac operon. When IPTG is added, the lac repressor is inactivated -> T7 polymerase and therefore the protein of interest will be expressed. The Lac I gene is already present in the genome of the expression strain used, why is it also cloned into the pET vectors?

According to this article, a single _lacI_ gene copy gives rise to about 10 copies of lacI protein per cell, and we can conclude, therefore, that this is the amount required to keep a single _lac_ operon repressed. The article also mentions the _lacI Q_ mutation in the promoter of _lacI_ that results in a ten-fold increase in the level of lacI protein.

If a _lac_ expression construct is present at more than 100 copies per cell the result will be leaky repression, and this can result in selection for mutations in the gene of interest if it has deleterious effects on the host bacteria. The easiest way around this problem is to put a _lacI_ (or _lacI Q_) gene on the expression plasmid so that repressor levels are commensurate with the number of copies of the _lac_ operator, as in the pET vectors.