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
On Sunday, August 18, 2013 1:55:45 AM UTC-7, Mega [Andreas Sturm] wrote:
On Sunday, August 18, 2013 1:55:45 AM UTC-7, Mega [Andreas Sturm] wrote:
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?
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