Dear all,
I am now working with a bacterial gene cluster and located 26 internal promoters within this cluster. Most of these promoters are found on coding region and I plan to remove them for fear that internal regulation might affect the protein expression later on in my project. Theoretically, promoters do not code for proteins but when they are found on coding regions, will excluding them affect the function of the expressed protein? I have thought of recoding the gene sequence to remove the internal promoters but by doing so will recode a new sequence not similar to the wild type. I want to exclude these promoters and leaves my protein unaffected. Are there any possible way of doing so? Thank you.
Dear Chee,
With only one promoter, efficient transcription through 14 genes sounds challenging. The natural promoters also probably play an important role in the stoichiometry of the various protein subunits. Do you expect the original promoters to be active in your expression system? Depending on your aims with the construct, maybe you could split the cluster in smaller parts so that you can at least have 2-3 promoters driving transcription of all the genes?
recoding the gene is not problem as far as i know, but removing the promoters codes from your operon i think it will affect on your protein structure and function. My advice you can synthesized your gene as peptide if it is possible and not so long.
How did you identify the promoters? And how many genes in the cluster? Why do you think these putative promoters will cause problems?
Dear Dr Elsayed, I would want to co-express the proteins after assembling the gene cluster in the cosmid so I think I will not be able to do as you suggested. I am thinking of recoding the sequence but one research team that recoded the sequence of nitrogenase gene cluster, had 2 folds reduction in nitrogenase activity. My concern is that I want to produce the protein in large scale.
Dear Dr Eldholm, I identified the promoters using a promoter prediction software and there are 14 genes in the cluster with two flanking genes. In my opinion, I will only drive the protein expression using the promoter on my expression vector and the proteins synthesized by the some genes in the cluster might become the transcription factors for other internal promoters in the cluster, hence interfering the expression driven by the external promoter.
Dear Chee,
With only one promoter, efficient transcription through 14 genes sounds challenging. The natural promoters also probably play an important role in the stoichiometry of the various protein subunits. Do you expect the original promoters to be active in your expression system? Depending on your aims with the construct, maybe you could split the cluster in smaller parts so that you can at least have 2-3 promoters driving transcription of all the genes?
Dear Dr Eldholm,
Thank you for the constructive advice. I think this is a good idea and will discuss further with my supervisor.
If you do want to get rid of the internal promoter sequences in your cluster coding regions you may want to do "silence mutation" as the 3rd base of a triple codon can be altered without change the coding aa so you can alter promoter seqs that way. using site-drected mutagenesis techniques. Could be a bit tedious then you can compare with your "wild-type" see which would be better. I bet you don't even need to worry about those "internal promoter seqs" because they could be "potential" as predicted by your program or just the "pseudo" seqs which may not be functional. Good luck with your experiments.
This is going to be very difficult. If you want to port this entire system to E. coli or another host for recombinant expression... Something is going to go wrong somewhere. Probably multiple things are going to go wrong at the same time, and then you don't know where to even start troubleshooting. So I'm going to side with Vegard -- I think you want to break this problem down into manageable components, troubleshoot each one individually, and worry about putting them all together into a production system later...
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