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ascacioc, on 23 August 2012 - 01:43 PM, said:
wow...quite a lot happened here while I was in the lab. Just a few words to the first post after mine:
somatic hypermutation is epPCR: in the lymphoid system the mutation rate is 10^6 higher than in the normal cells; epPCR is amplification with a high mutation rate. And this is not semantics.
I give you that at least: indeed VDJ recombination is a totally different system than DNA shuffling....even though parallels can be drawn.
Both DNA shuffling and epPCR are very controlled: now, if you use genome shuffling a la Stamer (first protocol ever published) you indeed cannot control it, but recent protocols for both epPCR and DNA shuffling are very well characterized and you can predict what you have in the end in the test tubes. There are programs and algorithms/scripts that do that for you. I worked developing some myself during my master thesis...and there were unexpectidily quite good in telling me what some other people get in their test tubes which means that we did not have so many uncontrolable stuff happening in the test tube (as I thought in the beginning of my masters when I was like you: riiiight you can control it)
I did not link DNA shuffling and epPCR more than the basic two protocols used in directed evolution. They are totally differently.
Of course that what we call directed evolution in the tube would not happen in nature: depends on where the selective pressure lays. I mean: if I evolve for example glucose oxidase to be more active to use it for a biofuel (real project on which people are actually working) you will not get the same things as in nature because while you are lowering the Km and making the kcat higher by directed evolution for you purpose, maybe in real life it is not good for this enzyme to use up in a fraction of second all your glucose in an organism and release H2O2 like tons of it in the same fraction of second because the organism will starve and will be killed by the toxicity immediatelly. However, it matter where you put the selection pressure: if you choose a smart selection pressure to keep the good mutations that are beneficial for an organism, you can simulate evolution.
And about not agreeing with that sentence: well, you are not agreeing with an entire field.
@prabhubct: fast-backwarding: I do not know how the selection pressure would work to fast backward something. I only know how to improve stuff, not how to make them worse
Andreea
somatic hypermutation is epPCR: in the lymphoid system the mutation rate is 10^6 higher than in the normal cells; epPCR is amplification with a high mutation rate. And this is not semantics.
I give you that at least: indeed VDJ recombination is a totally different system than DNA shuffling....even though parallels can be drawn.
Both DNA shuffling and epPCR are very controlled: now, if you use genome shuffling a la Stamer (first protocol ever published) you indeed cannot control it, but recent protocols for both epPCR and DNA shuffling are very well characterized and you can predict what you have in the end in the test tubes. There are programs and algorithms/scripts that do that for you. I worked developing some myself during my master thesis...and there were unexpectidily quite good in telling me what some other people get in their test tubes which means that we did not have so many uncontrolable stuff happening in the test tube (as I thought in the beginning of my masters when I was like you: riiiight you can control it)
I did not link DNA shuffling and epPCR more than the basic two protocols used in directed evolution. They are totally differently.
Of course that what we call directed evolution in the tube would not happen in nature: depends on where the selective pressure lays. I mean: if I evolve for example glucose oxidase to be more active to use it for a biofuel (real project on which people are actually working) you will not get the same things as in nature because while you are lowering the Km and making the kcat higher by directed evolution for you purpose, maybe in real life it is not good for this enzyme to use up in a fraction of second all your glucose in an organism and release H2O2 like tons of it in the same fraction of second because the organism will starve and will be killed by the toxicity immediatelly. However, it matter where you put the selection pressure: if you choose a smart selection pressure to keep the good mutations that are beneficial for an organism, you can simulate evolution.
And about not agreeing with that sentence: well, you are not agreeing with an entire field.
@prabhubct: fast-backwarding: I do not know how the selection pressure would work to fast backward something. I only know how to improve stuff, not how to make them worse
Andreea
you are right about, my mistake.
Quote
somatic hypermutation is epPCR: in the lymphoid system the mutation rate is 10^6 higher than in the normal cells; epPCR is amplification with a high mutation rate. And this is not semantics
Its indeed as you said: after binding antigens this starts, but the receptors themsellf (the first ones) are allready made at that time. I was thinking you ment the entire proces from the start which is of course not right.
PS.: you state that I am not agreeing with an entire field, I dont know if you are an immunologist, but if you are you, should know that even they arent agreeing completely, a lot of questionmarks are still out ther in that specific field. Its a pretty new field. Altough they do seem to agree on the proces, but how it happens exactly etc... still a debate going on.
about the followig:
Quote
Both DNA shuffling and epPCR are very controlled: now, if you use genome shuffling a la Stamer (first protocol ever published) you indeed cannot control it, but recent protocols for both epPCR and DNA shuffling are very well characterized and you can predict what you have in the end in the test tubes. There are programs and algorithms/scripts that do that for you. I worked developing some myself during my master thesis...and there were unexpectidily quite good in telling me what some other people get in their test tubes which means that we did not have so many uncontrolable stuff happening in the test tube (as I thought in the beginning of my masters when I was like you: riiiight you can control it)
This is just it: either you control it and then you are not really working with evolution, because evolution is not really controlled at all.. (at least not at the level people control it in test tubes during this kind of experiments). Or you cant control it and you do a completely random shuffling and then there is no link with evolution at all because you shuffle it way more then nature.
The thing is: you can control it because you set certain values yourself at the start! You start controling it yourself ..... thats not what you call evolution.
You pick the strains, you pick enzymes (sometimes, or more often then not, they work with restriction enzymes not even natural to the organisms), you pick the working temp, the media, ... you control a lot of the parameters...
BTW: the entire discussion here is nothing more then what physicist (and biologists) are debating for yours: chaostheory, randomness etc
Or on a footnot: what religious people are also stating.
Also, I like what you said about "evolving", but this sentence kinda breaks it down:
Quote
However, it matter where you put the selection pressure: if you choose a smart selection pressure to keep the good mutations that are beneficial for an organism, you can simulate evolution.
A lot of the work done on micro-organisms in the field of "evolution" is not really following the debate about what evolution now really is. The definition of evolution is changing almost every 10 years and even when people (like you?) speak about fast foward evolution in bacteria/yeast, we arent even able to correctly link protists based on evolutionary mutations etc...
All we do in the lab is cause mutations to occur , have genes rearranged , check for "better" organisms we can use or "new" organims and then we call this evolution. There are papers out there describing the "evolution" of a bacterium towards a better bacterium to bio-degrade some toxicA, is this really evolution? Is putting genes from yeast 1 in bacterium 4 and then caling this evolution really correct?
Dont forget that in many shuffeling experiments you mix genes from different yeasts ....
Or you push the organisms towards a certain evolution.. like you allready said: you select based on what you (we) want... weird view of evolution to be honest.
Your sentence said it, it makes my point:
Quote
@prabhubct: fast-backwarding: I do not know how the selection pressure would work to fast backward something. I only know how to improve stuff, not how to make them worse
what we call improving is based on our needs, and the definition of making things worse is also based on our standards. Dont forget that in the history of evolution a lot of the so called "bad" evolutions turned out to be good.
Evolution seems to be a very wide definition for many biotechnologists.
I think there should be some new definition or general agreement on what we do: evolve things like we want vs (real) evolution.
