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Background:

In synthetic biology, and also in nature, there are lots of examples of genes in both the forward and reverse orientation. It seems in synthetic biology/bioengineering, most genetic devices are, by default, encoded on the forward strand unless there are concerns of readthrough from one expression unit to another, in which case one of the expression units is included on the reverse strand like below:

enter image description here

It also seems that genes are sometimes in the reverse orientation for no apparent reason, especially in plasmid backbones:

enter image description here

The Question:

My question is, ignoring issues of potential readthrough, does it matter which strand a genetic device is encoded on? So in the example below, would there be any difference between A and B, or does it not matter? Also, does whether it's on a plasmid compared to a genome matter? I'm specifically interested in bacteria here.

enter image description here

This seems like it should be a simple question to answer and I'm sure I'm just missing something, but I can't find anything about it.

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    $\begingroup$ I would consider your terminology very carefully, because although your concern is reasonable, the question misuses scientific expressions in a manner that hinders communication. First, genes encode RNA and protein: neither they nor constructs are encoded by anything. Second, each gene has a forward and reverse strand, but almost no genomes do. So what you are talking about is the relative orientation of genes or constructs within a genome, plasmid etc. It would be good if you could reword your question accordingly. $\endgroup$ – David Apr 2 at 13:07
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    $\begingroup$ Thanks for the comment, I'll edit based on your first point. For the second point, I think that my incorrect use of terminology for forward and reverse is what caused me to ask the question in the first place, because when I tried to correct them there was no longer a question, and this was also mentioned in the answer, so maybe it's best to leave that in the question so the answer still makes sense? $\endgroup$ – Brad0440 Apr 6 at 8:19
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First, I think you have misunderstood the concept of sense/antisense. These are defined individually for each gene, with the sense strand always referring to the coding (non-template) strand of a gene, and the antisense strand to its complement, the non-coding (template) strand. So in your plasmid example above, all genes are encoded by their respective sense strand, but which of the two strands that is sense for a given gene depends on its orientation. There is therefore no biologically meaningful sense/antisense concept for entire genomes, chromosomes or plasmids. Read more in the Wikipedia article on sense, and the answers to this SE Biology question.

Having said that, it should be clear that the expression cassettes pictured in figues A) and B) are completely identical. If you ignore the context around them (e.g. readthrough), their direction is irrelevant.

It also seems that genes are sometimes encoded on the reverse strand for no apparent reason, especially in plasmid backbones

Many commonly used plasmid backbones derive from vectors that date many decades back, to a time when cloning strategies were far from as flexible as today. Researchers were generally limited by the restriction sites occurring naturally in the DNA sequences. A gene's position and orientation with respect to its neighbouring genes might therefore be the simple result of lack of other cloning options - although I honestly doubt "wrong" orientation would even have been considered an issue. For an example you could read about plasmid pBR322 in this Wikipedia article, and references therein.

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    $\begingroup$ Ah yes, of course, I thought I was missing something silly! Thanks for the answer! $\endgroup$ – Brad0440 Apr 2 at 7:35

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