In Bacillus subtilis, there is a strong orientation bias so that DNA replication and transcription are co-directional (see the reference below). We think that the genomes are arranged to avoid collisions between the replication and transcription complexes. I agree with Andrew that the difference in expression levels may be negligible for a given gene. But since you have the option of orienting your insert whichever way you want, why not make it co-directional with amyE? Also, keep in mind that most common amyE integration vectors require you to supply a promoter for the cloned gene, and that a Gram-positive promoter is required in B. subtilis because of the strict sequence requirements of the Shine-Delgarno box. But amyE integration is a standard technique in the B. subtilis genetics arsental. You can do it. :)

http://www.ncbi.nlm.nih.gov/pubmed/2118869

Everything will depend if the promoter is part of your gene or not.

Hi Daniel,

I am not sure about chromosomes, but when you insert a gene into a plasmid you don´t really care about the orientation. As far as the gene has its own promoter it should be expressed independently of the orientation of flanking region. Chromosomes are more sofisticated structure than plasmids but I think the orientation should not affect your experiment.  What I would be really careful about is to insert the gene in a euchromatic region…

It doesn't matter as long as the gene of interest in an expression cassettee:  Promoter+Your gene+Terminator region

Hi Daniel

Since we know expression of genes in eukaryotes is complicated with transcription initiation and chromatin modifications, I think orientation of gene do matters. However you are interested in inserting a gene into a bacterial chromosome which is little less complicated than eukaryotes and since it has its own promoter there may not be a problem with orientation. So give a trial and see how it behaves

All the best

Hi Daniel,

It does not matter if you are planning to insert the complete gene

Hi Daniel

Orientation of the Gene does matter. If you desire correct expression of gene (and a functional expression of the same), the gene and promotor needs to be inserted in the 3'-5' strand cause thats the one read by bacterial RNA-Pol and the gene must be inserted that way. If the gene is put in the genome in a reverse order, transcription will not occur. Actually, that is something that must be always planned when constructing your plasmid so it does have the gene inserted in a correct way so you proceed to bacterial/yeast/microrganism transformation/gene complementation. For that, normally, when you amplify your DNA fragment by PCR you design  your primers in a way they will create complementary ends with your plasmid - such ends being different in each cut point, that was originated by the cut of, usually, 2 different restriction enzymes.

I hope that helps.  

That depends on

That covers the basic questions one would need information on on order to answer your question. Otherwise, it would just be guesswork.

Perhaps. You might see a difference for a few reasons: upstream/downstream genes have relatively strong/weak transcription, upstream/downstream sequences vary with respect to supercoiling, and a higher likelihood of premature transcription termination if transcription of your gene collides with the replication fork.

Probably it won't make a big difference, but it's good to be concerned about it.

I am working on B subtilis.  I want to put a whole gene cassette (promoter-gene-terminator) into an integration vector so that my cassette is flanked by amyE gene that I am going to insert into.  I am not sure whether my cassette should be on leading strand of amyE or the opposite so that my gene could be expressed.  

As you are working with Bacillus, directionality might indeed matter. I have just checked amyE in that genome and it is indeed located on the leading strand. So your cassette should point in the same direction as amyE.

If you want to avoid transcription of the 5'-part of the then split amyE leaking into your construct, you also might consider putting a terminator upstream of your promoter, so that the whole thing inserted into the chromosome looks something like amyE'::T::Pgoi::goi>::T::'amyE>

Hello there,

opinions differ and my guess would be that you could clone it in both directions and screen for expression. I am more experienced with cloning in plasmids so if you clone in both directions and also in the leading strand that may give you an answer that you can use in your future work too.

It is more laborious (having to try everything) but you will be sure in the end of what works best for you.

Best wishes,

Nikos

In Bacillus subtilis, there is a strong orientation bias so that DNA replication and transcription are co-directional (see the reference below). We think that the genomes are arranged to avoid collisions between the replication and transcription complexes. I agree with Andrew that the difference in expression levels may be negligible for a given gene. But since you have the option of orienting your insert whichever way you want, why not make it co-directional with amyE? Also, keep in mind that most common amyE integration vectors require you to supply a promoter for the cloned gene, and that a Gram-positive promoter is required in B. subtilis because of the strict sequence requirements of the Shine-Delgarno box. But amyE integration is a standard technique in the B. subtilis genetics arsental. You can do it. :)

http://www.ncbi.nlm.nih.gov/pubmed/2118869

Actually it does not matter. However, what type of vector you plan to use? I would normally use a blunt end vector for this purpose and not one with open sticky ends.