In Vitro Evolution - (Feb/08/2009 )

Hello peeps,

I am not too sure if this is the right place to post this topic. But I am working on In Vitro Evolution and was thinking of starting a discussion for it since it is related to evolution, but within the lab.

Tell us about your research.

GeorgeWolff on Feb 9 2009, 06:41 PM said:

well, i am trying to make mutated libraries so i can use in vitro evolution to evolve the protein that i am working on. I am unable to list down the things i am working on right now because of confidentiality issues by the institute. was just thinking of whether anybody has done anything related to directed evolution.

Directed evolution - understand you can't report the application but can you tell us some of your methods - e.g. how are you selecting for modifications? Also, please consider that proteins do not evolve.

jiajia1987 on Feb 9 2009, 03:36 AM said:

I was speaking to someone who was working on something like this, I think, where they were testing the effects of mutations of a ligand, trying to increase its affinity for its receptor (with pleasing results, if I recall). I don't recall whether they were reasoned mutations or a large library. Can you call this in vitro evolution or do you mean something else?

I imagine you can also stimulate evolution in a particular direction for example, if you have a bacterial cell reliant on certain conditions, and you vary the conditions slightly towards an abnormal but desired state. If you keep changing the conditions slightly, you may be able to produce proteins capable of working in that particular environment. Of course it depends on what the condition is (heat would pervade the bacteria) whereas antibiotics might just be pumped out without inducing antibiotic-reistant enzymes (i.e. resistance to antibiotics targeting folic acid synthesis could come about by evolution of many things other than those enzymes directly involved in synthesis).

Yes, this is exactly what I mean by in vitro evolution, or perhaps we could be more precise by calling it directed evolution since we are 'directing' the evolution to the direction we want it to go, but it isn't an easy task.

I replied earlier to georgewolff once about what I am doing, with the details, except that the protein names were hidden. But I deleted the post later on due to confidentiality issues. I am definitely glad that someone like you posted to this topic!

Hi,

I'm working in the field of directed evolution as well. In particular torward selection of protein and RNA from completely random libraries to assess whether and to what extend random biopolymers may display catalytic properties.

I notice the post of GeorgeWolff who stressed that proteins do not evolve. I usually deal with a library of random protein (that can be regarded as a population) which is selected for a desired function by means of iterative cycles of selection-amplification-mutation so that the final protein library (or population) has distinctive features (sequence, composition, structure). Isn't it evolution on a much simpler scale?


Cheers,
dldavide

I've done some directed evolution. The critical part is how you do the selection. Anyone can make a bunch of randomized DNA sequences.

dldavide on Aug 21 2009, 11:00 PM said:

this is precisely what i do too. i deal with a mutated library of a particular gene, which undergoes in vitro translation in the process of selection to form mutated versions of the protein that i want and see which protein has the desired property.

Phaedrus on Aug 22 2009, 05:28 AM said:

it's true that anyone can make a bunch of randomized DNA sequences, but the randomization part is kinda critical too. it's not the quantity that counts, but the quality of the randomization, right?