for heterologous expression in B.subtilis, I have used a triple promoter for expression of 2 genes in one vector , each gene has its own RBS, but there is only one triple promoter before gene 1, and then RBS for gene 2 and a terminator at the end, is it possible that expression of gene 2 decreases because it dos not have a separate promoter? and for high efficiency should I add a separate promoter before gene 2?
thank you
Assuming you don't have a large space between the end of gene 1 and the start of gene 2, then a single promoter should be fine. In my experience whether a gene is well expressed or not is more often dependent upon either turnover (degradation) or inefficient translation initiation and not so much on high level transcription.
Single promoter is fine and I also clone the smaller gene downstream of the larger gene.
Michael J. Benedik Dear Dr Benedik thanks for sharing your experience and your help, the space between 2 genes is short ,but I am afraid that ribosome does not attach to the RBS of the second gene and the transcription of the second gene decrease , is it a possibility ?
Aekagra Singh Thanks for your response, but the first gene is not a native gene for bacillus , but second is a native gene and the size of both genes are approximately equal about 1300 bp . does it work in your experience ?
regards
one more question in some vector the direction of the genes are forward and in others it is reverse ( genes located behind the promoter) ,does it work if I use reverse direction for my genes on my expression vector? the vector integrate to bacterial chromosome.
Yes it can work just make sure the promoter is bidirectional for opposite direction genes and since each gene has its own RBS then it's even better. There are some polycistronic vectors that I have come across that use a strong promoter to drive expression of 3-5 subsequently cloned genes each having their own RBS, so 2 genes should not be a problem. Just make sure if the genes are running in the same direction their is no transcriptional terminator site or stretches of low GC content in the intergenic regions otherwise the polymerase might terminate transcription.
Nazanin Mehrzad the ribosomes should bind to the RBS of each gene that you have. This is the underlying premise behind how operons work in bacteria.
Regarind your second question about directionality, I don't think the answer is simple. First of all, all the genes should be in the same orientation as the promoter providing transcription. So directionality is important here. A bidirectional promoter would help if gene 1 is pointing left and gene 2 is pointing right and you clone the promoter inbetween. But it doesn't sound as if you are doing that.
If the plasmid is built correctly then upon integration the directionality should be conserved (assuming that I am understanding your experiment correctly).
Michael J. Benedik Dear Dr Benedik ,Thanks for your explanation , for clarifying this ,I reverse all construction including promoter , and both genes ,I mean I oriented them in anticlockwise on my vector, as I saw on some articles that they use anticlockwise orientation on their vector. does it work in your experience ?
regards
No, it should be totally irrelevant, unless there are other elements on your plasmid that might interfere. Clockwise or anti clockwise is an arbitrary distinction.
Michael J. Benedik really appreciate your answer, do the restriction enzymes in my vector matter? as you said other elements may be exist.
regards
by other elements I meant strong promoters or such. Restriction sites don’t matter except as they are used for your cloning.
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