[pazimzadeh]

What is the doubling time for the E. coli you are working with?

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.

[koeng]

Normal E.coli doubling time, which means about half the speed of Vibrio natriegens. I use NEB turbos sometimes, but they still don't bring down the liquid culture time down from overnight to within a day, mostly.

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.

[pazimzadeh]

If all you're interested is cloning/expression, then I guess that makes sense, although I've not heard anyone complain that 20 min is a slow doubling time. can't you just inoculate your starting culture with a larger amount or something? I often find myself starting cultures late in the day so that they don't overgrow the next day (although I am not doing expression work).

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?

[koeng]

> can't you just inoculate your starting culture with a larger amount or something

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.