We need to construct orpon of 6 tandem genes (total 5 kbp) for protein expression in E.coli. We have previously constructed this opron using gnomic DNA and subcloning but we were not pleased with protein expression. So, we have decided to try codon optimization and gene synthesis.
Would you think single gene synthesis and subcloning is a better option or whole opron synthesis ? all we care is protein expression of all six genes in opron and not single expression.
Thanks!
Hi Mahmoud,
That's quite an expression project!
Some thoughts:
To generate the whole operon through gene synthesis will almost certainly be done by building it up from smaller components of say 500-1000bps, which could be considered as each of the component genes if you desire it. How the company you use joins these components is up to them, they might use restriction digest or a PCR based overlap extension. You can have restriction sites incorporated where you want however so it doesn't really mater. This means that you can have both the individual genes generated and a full operon of the 6 genes made if you design it right. There is no reason not to have the component parts delivered as well as the final construct.
I would recommend trying to incorporate as much flexibility into the design, as it is hard to judge what further issues there will be with the expression of the individual genes. so being able to remove a gene, or change the order on the operon would be useful. Talk to the people doing the synthesis, they must have had similar requests in the past and have some good ideas.
You call them tandem genes, if there is a high sequence similarity between them, then I would suggest trying to reduce this as far as possible at the sequence level. This should help with DNA stability of the final construct.
I would also suggest that you make sure that all the genes have an optimised RBS site.
-Richard
Thanks Richard for the comment. Yes, I am asking them for their opinion too. However, none of the gene synthesis providers are guaranteeing protein expression from constructed plasmid which make me worry to waste money, is there any particular vendor you prefer ?
In our lab we usually use SpeI/XabI compatible end to create orpons. XbaI in pET vectors provide us with the RBS. Therefore, the final opron just has one promoter. Would you think it might help protein expression if provide each gene with a independent promoter ? Talking bout an optimized RBS, we use strong RBS in pET28a as our default choice. Would you suggest particular RBS optimization process?
Thanks
Hi Mahmoud,
I wouldn't expect any company to guarantee expression, there's just too many reasons for it not to work that have nothing to do with the DNA sequence. As for companies it's been a few years since I've ordered a gene however I've used GeneArt (now part of ThermoFisher) most and found them very good and knowledgeable, even questioning what I have designed at times to check that what I had ordered was really what I wanted. I've also used GenScript & DNA2.0 before with success.
The RBS from pET is a good one, it would need to copied to the start of each gene in the operon, for good expression from each gene.
I really don't know if multiple promoters would be a good idea or not. My inclination is not. Maybe 2 could work, so 3 genes each.
Are you wanting these proteins to generate a complex? One thing that might be required if so is to have the worst expressed gene on a separate plasmid (with a compatible origin) to see if you can generate more, or to use a different inducer.
I expect that you are entering an iterative process here, where each step will, hopefully, make a positive result. It is however hard to second guess what will be the best steps to take.
Good luck
-Richard
Yes, I hope to generate a complex of these proteins. Thanks for your time and help.
Mahmoud
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