Hi Liang,

It depends on the organism / strain that you are using for the expression, because the lacI repressor is not encoded on the plasmid. Does your strain express the LacI repressor? If yes, you could probably enhance the expression of your target gene by induction with IPTG. If not, transcription from the lac promotor is not repressed and thus cannot be further induced.

Have you checked the complete insert/plasmid by sequencing? Maybe your complementation does not work because of a mutation.

Best regards,

Frederik

Hi Frederik Walter,

Thanks for your rely. Yes I' m sure that the LacI exists in my strain which is a clinical isolate. So I'll follow your suggestion.  

I'm doing plasmid complement test. The phenotype ( resistance for antibiotic) decreased drastically  after knockout of the target, however, the resistance resotred to half of the original strain by plasmid complement. I'm looking for the reason for this, or this is a common phenomenon since the change of gene's environment.   

So, I think maybe the resistance is not fully restored  since the expression level is lower than the gene in gnome DNA.

Best,

lac promoter is more active and inducible than the gene promoter you cloned. It is optimised for the plasmid. Since the vector has it own rbs, the ribosomes will bind to the vector rbs in the lac initiated trascripts and search the ATG for protein translation. Your protein ATG is too far away for the vector rbs (due to your gene promoter) so most ribosomes will fall off from the mRNA that causes the protein translation is lower that you expected.

 Hi Dr. Liu,

Thanks for your reply. I noticed the Lac promoter is too far to indcuce the transription of my gene. So as you mentioned , my gene will be dificult to be induced. 

Besides that, I have a question:

I'm doing plasmid complement test. The phenotype ( resistance for antibiotic) decreased drastically  after knockout of the target, however, the resistance resotred to half of the original strain by plasmid complement. I'm looking for the reason for this, or this is a common phenomenon since the change of gene's environment.   

Best,

Huang Liang 

This is quite common in the complement test, you think that you have cloned the full promoter of your gene, but it is quite difficulty to prove that, moreover due to the environment changes, even the full promoter may failed to functions as it is in the genome. It is acceptable that you can do the complement analysis just use the promoter of the vector to drive you gene expression, in that case you might be able to restore to the wild phenotype.

Hi Liang,

Only partial phenotype restoration by complementation is not uncommon in bacteria. The importance of DNA topology in bacterial gene expression is more and more documented and might of course be very different on the plasmid as compared to at the "natural" location in the chromosome.

See for example :

DNA structure and function

By: Travers, Andrew; Muskhelishvili, Georgi

FEBS JOURNAL Volume: 282 Issue: 12 Pages: 2279-2295 Published: JUN 2015