Everything You Ever Wanted to Know about E. Coli (2008)
scientificamerican.com
scientificamerican.com
We absolutely do not have a clear idea of how E.coli works. Hell, we don't even know how almost 1/3 of the genes work on JCVI-Syn3a works, a minimal genome we synthetically created. Far fewer in E.coli.
E. coli will never form a spore (unless you engineer that somehow?), why would you want E. coli to sporulate? Some of the things you mention can be done by clinical strains. Couldn't you just make the E115K purB mutation if you want that? I agree that lambda red is limited.
I would want E.coli to sporulate because then shipping and distribution of strains is a lot easier. This was a problem back when I was shipping at FreeGenes and still is a problem. I used Bacillus subtilis to make the sporenet protocol which worked, but the vector backbone switching was pretty painful.
Clinical strains == not necessarily GRAS. Also, they're clinical, so regulated by MTAs, which are a big no-no.
I could make the E11K purB mutation, but why would I go through the effort in an inferior species of bacteria when I can invest my time into something like Vibrio natriegens? The payoff is better at the end.
Vibrio natriegens seems great for your purposes. I'm curious if there is a tradeoff to its super fast growth. Does it have a greater mutation rate?
No, I'm doing high throughput cloning in such a way where a limiting factor is going from 1 cell to X cells (usually pickable colonies). So liquid culture doesn't matter quite as much, since I can sequence validate from a colony split (half into colony PCR, half into new culture), and sequencing takes approximately as much time as the growth step takes.
> I'm curious if there is a tradeoff to its super fast growth. Does it have a greater mutation rate?
Eh, not really. The biggest tradeoff is that it isn't studied nearly as much as E.coli, so the chemical competence protocols kind of suck. I can't actually use it in my pipeline right now because of that. So I'm stuck with E.coli until I can figure out a good transformation protocol.
My side would immortalize b-cells with Epstein-Barr... but that experience left me with a healthy respect for e coli.
Even just within the subset of E. coli which causes UTI's, 25-40% of the genome varied between strains. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653229/)
This diversity wasn't really appreciated in 2008 when many E. coli genomes hadn't been sequenced yet.
It turns out that the Escherichia coli (to spell out its Latin binomial) that cause disease are in some sense “diseased” themselves: the genes that enable them to be pathogenic, or make them pathogenic, I should say, are originally from a phage, a type of virus that infects bacteria [1]. In a manner that is not the same as, but conceptually similar to how HIV inserts its genes into the human’s genome, phages insert their genes (termed the “prophage”) into the bacterial genome.
In addition, most strains of pathogenic Escherichia are also holding on to an entirely separate, small, circular “genome” called a plasmid, also of exogenous origin, that contains additional genes that make them pathogenic.
So in addition to wide genome variation within the “species” (which is not really the same thing for bacteria as for mammals, mind you) of Escherichia coli, many subtypes have additional genetic material from endogenous sources that substantially changes their observed characteristics (phenotype).
Do you have a citation on the fact that 'most' pathogenic strains have a plasmid making them so? Some guys in our lab have been playing around with plasmid copy number lately (in a largely 'basic science' kind of way) -- this could give some nice context for that work.