I knocked out my target gene, in order to confirm its function, I did the plasmid complement test.
I cloned the gene with its full promoter into the MCS, however, it seems that the expression level of this gene is not enough to restored the phenotype. So, I decided to use IPTG to enhance the expression.
My question is: Is the Lac promoter inducible if the cloned gene has its own promoter ?
Or there is something wrong. I used the pCR-bluntII-topo
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
Yes, the lac promoter will be induced. However, as the others have pointed out, the actual expression level will depend on the length and structure of the RNA sequence between the lac promoter and the gene you want to express.
Note that if you don't have lacI, the lac promoter will be expressed constitutively.
Don't forget that the lac operon is also under catabolite repression, so you should make sure that the medium doesn't contain glucose as this will restrict lac expression.
It would be helpful to know what organism you're working in and whether your gene is on a high copy plasmid, low copy plasmid, phage or something else.
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