3
$\begingroup$

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?

Improve this question
$\endgroup$
4
  • $\begingroup$ Because you can then use this system in bacterial strains which do not have LacI. $\endgroup$
    – Chris ♦
    Commented Aug 4, 2014 at 15:28
  • $\begingroup$ Thanks for you comment @Chris. Are there strains that have the T7 RNA polymerase but do not have lacI? Or are those then just infected with T7 phage to express the protein? Could you find a reference for your statement and post it as an answer? $\endgroup$
    – jan-glx
    Commented Aug 4, 2014 at 15:59
  • $\begingroup$ I don'T know about all strains - sorry. But you could co-express it from another plasmid. $\endgroup$
    – Chris ♦
    Commented Aug 4, 2014 at 16:05
  • 1
    $\begingroup$ Another possibility (and probably more likely) is that the construct is less leaky that way. $\endgroup$
    – Chris ♦
    Commented Aug 4, 2014 at 16:11

1 Answer 1

Reset to default
4
$\begingroup$

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 lacIQ 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 lacIQ) 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.

Improve this answer
$\endgroup$

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .