DIY targeted evolution

[Mega [Andreas Sturm]]

Hi everyone,

I was just thinking the following:

Salpetric acid causes mutations (it converts T -> U, then it's replicated error-pronely)

http://de.wikipedia.org/wiki/Pyrimidine#Abbau_und_Modifizierung_der_Basen


So if I keep the E. Coli with the glowing plasmid on LB-Amp-agar containing HNO2, they will mutate. At some time, ther may occur a transformant that glows yellow or purple or red or pink. That would be nice. Of course, for each "positive" transformant there will be hundreds that will have metabolic knock-outs and will die. And many which switch off the light (some fatal mutation in the luciferase)


Does that sound feasible?

How much HNO2 would you add? to pH 5.4?  Or just very low?

[Mega [Andreas Sturm]]

Ah sry, english languge got me again. Or actually, German language, because our nomiclature is strange.

The term for HNO2 is Nitrous acid.

[Cory Tobin]

I'm not sure I understand why you need nitrous acid, but just FYI,
nitrous acid can only be made in situ (you can't buy a bottle of
nitrous acid). You make it in solution by mixing sodium nitrite and
HCl in an ice bath. Also, the nitrous acid only persists in solution
at temperatures close to 0C. Once you heat it up to room temperature
it will decompose into some nasty gases.

Also, a long time ago I was doing some diazonium chemistry (diazoniums
are made with nitrous acid) and I think I remember there being a
maximum concentration that nitrous acid can be at before it
decomposes, even at 0C. I think it was something like 200mM. So
that's something to keep in mind.

If you're just trying to grow bacteria on mutation-inducing media I
would recommend ethyl methanesulfonate instead.


-cory

[Mega [Andreas Sturm]]

Ok, that should work as well. One could also incubate just the plasmid in hno2 and then transform. Should work even better as the entire colony would have the new trait then.

[Koeng]

Its kinda ironic, the lab I am working at is currently working on a system for directed evolution. I think they said that it is 10,000 times more effective then any other method. I think ima try GFP on this :)

How it works: 

In some yeast, there are linear plasmids in the cytoplasma, and each on holds its own, or another plasmids dna polymerase. These polymerases ONLY replicate the linear plasmids, aka they have a totally different replication system then the hosts. So what they are doing is integrating an extremely error prone polymerase (call it polymerase A) into the genomic dna. Then plasmid A, which is essentially blank, is extremely error prone. Then the user can integrate the DNA they want to use into the blank plasmid A. Then this plasmid will be replicated, and since it is extremely error prone, mutation will happen EXTREMELY quickly.

Any who, if you wanted to do directed evolution in bacteria, use a BAC. Right now I am working on getting the plasmid number in that system down to 1-5 since all the non-mutated ones will overwhelm the mutated ones if the plasmid copy number was high

Review on linear plasmids here

http://link.springer.com/chapter/10.1007%2F7171_2007_095

I know this probably doesn't help but oh well, maybe i gives you an idea

-Koeng

[Nathan McCorkle]

Sounds like you'd need single-cell analysis with an innate mutagen.

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[Mega [Andreas Sturm]]

Interesting...


But I guess it's gonna be like that:

I ask a chemist friend to produce HNO2 in situ. Then I incubate the plasmid in it and do a transformation.


Maybe that'll be sufficient to produce a lot of mutants....

[Josiah Zayner]

T->U will cause A to replace the U in transcription.
http://www.sciencedirect.com/science/article/pii/S1097276503003605

Things like alanine GCT->GCA causes no change and glycine GGT->GGA and many others will cause no change, 27 codons don't even have a T. You will mutate your start sites (ATG) and your top codons(T**). Your search space is so small using this technique because each codon will only have at most(TTT) 3 other possible codons it can become. So you will be able to try 1000s(10^3-10^5) of different constructs. Not a large search space. Most mutagenesis strategies search 10^9 - 10^12 sequence space and are limited by transformation efficiency.

Random mutagenesis has been done so much just look up papers.
A simple way is to make degenerate primers.
Or use a MutL or MutS deletion strain of bacteria.
or directed evolution
http://nar.oxfordjournals.org/content/28/16/e78.short

or many many other things.

http://scholar.google.com/scholar?hl=en&as_sdt=0,14&q=bacteria+random+mutagenesis

[Koeng]

I think they go for colonies, although I recently joined and don't have hands on with it yet

[Nathan McCorkle]

On Mon, Aug 19, 2013 at 11:29 AM, Koeng <koen...@gmail.com> wrote:
> I think they go for colonies, although I recently joined and don't have
> hands on with it yet

I don't see how the colony would have a clonal plasmid, since it's
potentially messing up X number of times per division (or maybe the
metric is number of divisions per substitution).