Dear all, I am currently working on a synthetic biology project where I have to construct my own plasmid for protein expression. In regards with protein expression, I have chosen the inducible T7 promoter and IPTG will be my inducer. However, is it necessary to include a LacI region downstream of the promoter? I understand that the repressor binds to the operator region on the T7 promoter, down regulating the transcription from the promoter and when IPTG is added, the IPTG mimics allolactose which changes the conformation of the repressor and allows DNA polymerase to bind to the promoter. In my point of view, I should include LacI as this will produce my repressor protein or otherwise, there will be constitutive transcription from my T7 promoter. Would appreciate constructive comments and advice. Thank you.
Hi there,
If you are hoping for a tight regulation of T7 promoter, cloning lacO sequence downstream is a good idea but as you mentioned it is advised to clone the lacI gene on the plasmid too in order have enough lacI to bind lacO in vivo...
Dear Dominique, thank you for your suggestion. After a second thought, I think that not including the lacI region is also possible. Supposing that the transformation host that Im using is an E. coli and the host itself has chromosomal genes encoding for T7 RNA polymerase, naturally the lacI is also present on the chromosome of the bacteria itself. Therefore, the lacI repressor from the host will naturally bind to the LacO.
I agree but the number of copies of LacI protein could not be enough to repress the lac promoter controling the expression of T7 RNA polymerase gene and the T7 promoter present on the plasmid especially is if the plasmid is high copy number... It's a matter of ratio between ligand and sites for this ligand. That's why most of the commercial plasmids containing a lacO site also encode for lacI.
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