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Deletion Mutant - Evolution - (Aug/19/2012 )

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ascacioc on Wed Aug 22 19:06:14 2012 said:


@Pito: genome shuffling happens in nature: V(D)J recombination is DNA shuffling; this is the way your body makes antibodies by combining the alleles in the MHC class I loci; moreover in the next step, your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus.

The point of epPCR is that it actually recreates the same most probable mutations/errors done by DNA polymerases in bacteria. There are tons of papers calculating the probabilities of certain transitions/transversions and the hot spots for mutations and they are pretty much the same no matter what polymerase you are using. So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube. (some people use this cliche over and over again in the directed evolution field)

@prabhubct: if you would like to read more about directed evolution: http://www.sesam-bio...ected-evolution A pretty good review of the state of the art 2-3 years ago (I wrote it:P) It also has references to material that paraallels directed evolution to natural evolution.

Andreea



@ asacioc as you said '' So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube.'' could it be fast- back-warding natural evolution?

-Inbox-

pito on Wed Aug 22 20:04:47 2012 said:


ascacioc on Wed Aug 22 19:06:14 2012 said:


@Pito: genome shuffling happens in nature: V(D)J recombination is DNA shuffling; this is the way your body makes antibodies by combining the alleles in the MHC class I loci; moreover in the next step, your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus.

The point of epPCR is that it actually recreates the same most probable mutations/errors done by DNA polymerases in bacteria. There are tons of papers calculating the probabilities of certain transitions/transversions and the hot spots for mutations and they are pretty much the same no matter what polymerase you are using. So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube. (some people use this cliche over and over again in the directed evolution field)

@prabhubct: if you would like to read more about directed evolution: http://www.sesam-bio...ected-evolution A pretty good review of the state of the art 2-3 years ago (I wrote it:P) It also has references to material that paraallels directed evolution to natural evolution.

Andreea


Of course genomeshuffeling happens in nature, but there is a difference between what you call genome shuffeling that happens in nature (there is a system behind it, I am talking about the VDJ recombination now) and genome shuffeling we do in the lab.

And I do not agree with what you state here, its a bit "risky" to state it like this: "your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus"
This is not entirely correct.
The body does not "evolve" like you state it.
The body has allready a bunch of antibodies present, by pure luck a few of those happen to bind the antigen, because they do, they will be favored and other that do not bind will not (or less) by enriched (recreated) by the body. This is called affinity maturation of antibodies. This happens because the B cells with the best binding receptors will bind the anitgen and will be selected (and survive/multiply and pass on their genes) because they are able to bind the follicular dendritic cells (those will bind the antigen, so the B cells bind indireclty) this is how affinity maturation happens and how "evolution" of antigens work. ANd yes, due to simple mutations in those cells, you will also create better cells in the end. But its bit different from just stating what you stated.


But at the start: its all a random proces, your body just "creates" random antibodies by VDJ recombination (random; but with a certain system).

This is completely different from the genome shuffling you are speaking of. In genome shuffling you cut DNA and create random new pieces of DNA by extending them again, ligating them.
+ genome shuffeling and then compare it with VDJ.. a bit weird esp since I was talking about bacteria, but perhaps I did not state this clear enough.
In bacteria there is no such thing as VDJ recombination.
And I dont like to link systems we use in bacteria/yeast for human or animal systems/genes.

Also: epPCR can indeed be used as a tool to study what happens with bacteria/yeast for example and you can indeed call it fast evolution, but it doesnt really represent 100% what happens in nature.
Its just a tool to cause mutation, nothing more.
ANd yes, you could state that those mutation (or some) would indeed also happen in nature , but nature is far more complex.

Also: linking genome shuffeling and epPCR is a bridge to far for me.
epPCR is much more controlled while genome shuffeling is (or can be) less controlled and you can get stranger results.
Altough, in the end its all about how you define certain stuff.

Also, and you said it yourself: its called "directed" evolution, thats just it: we (the researchers) direct evolution.. you cant simple say: aha, this is what would happen in nature.
Its a bit easy to state that.

What you create in the lab, I wouldnt call it evolution. I would call it: an observation of changes in DNA that cause a certain (observed/measured) effect, which in the end could indeed be a representation of a certain (possible) evolution.
You need to keep in mind that many of the so called "evolutions" caused by direct evolution techniques would not survive in nature or stay evolved like this because we want this evolution and keep in, while in nature the evolution might we "stupid" and not wanted and thus be lost in the end.

But this is more about semantics.

But do not agree with "directed evolution is defined as fast-forwarding natural evolution in a test tube" because for me this is not correct. A lot of the so called "direct evolution" are no evolutions that would happen in nature.


thanks.
@ pito : as you said ''epPCR can indeed be used as a tool to study what happens with bacteria/yeast for example and you can indeed call it fast evolution, but it doesnt really represent 100% what happens in nature..'' i agree with you as directed evolution could not represent 100% of nature. But it do represent some probabilistic value for evolution if we do want to believe in transition, transversion, recombination as means of evolution.

-Inbox-

prabhubct on Thu Aug 23 05:58:08 2012 said:


pito on Wed Aug 22 20:04:47 2012 said:


ascacioc on Wed Aug 22 19:06:14 2012 said:


@Pito: genome shuffling happens in nature: V(D)J recombination is DNA shuffling; this is the way your body makes antibodies by combining the alleles in the MHC class I loci; moreover in the next step, your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus.

The point of epPCR is that it actually recreates the same most probable mutations/errors done by DNA polymerases in bacteria. There are tons of papers calculating the probabilities of certain transitions/transversions and the hot spots for mutations and they are pretty much the same no matter what polymerase you are using. So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube. (some people use this cliche over and over again in the directed evolution field)

@prabhubct: if you would like to read more about directed evolution: http://www.sesam-bio...ected-evolution A pretty good review of the state of the art 2-3 years ago (I wrote it:P) It also has references to material that paraallels directed evolution to natural evolution.

Andreea


Of course genomeshuffeling happens in nature, but there is a difference between what you call genome shuffeling that happens in nature (there is a system behind it, I am talking about the VDJ recombination now) and genome shuffeling we do in the lab.

And I do not agree with what you state here, its a bit "risky" to state it like this: "your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus"
This is not entirely correct.
The body does not "evolve" like you state it.
The body has allready a bunch of antibodies present, by pure luck a few of those happen to bind the antigen, because they do, they will be favored and other that do not bind will not (or less) by enriched (recreated) by the body. This is called affinity maturation of antibodies. This happens because the B cells with the best binding receptors will bind the anitgen and will be selected (and survive/multiply and pass on their genes) because they are able to bind the follicular dendritic cells (those will bind the antigen, so the B cells bind indireclty) this is how affinity maturation happens and how "evolution" of antigens work. ANd yes, due to simple mutations in those cells, you will also create better cells in the end. But its bit different from just stating what you stated.


But at the start: its all a random proces, your body just "creates" random antibodies by VDJ recombination (random; but with a certain system).

This is completely different from the genome shuffling you are speaking of. In genome shuffling you cut DNA and create random new pieces of DNA by extending them again, ligating them.
+ genome shuffeling and then compare it with VDJ.. a bit weird esp since I was talking about bacteria, but perhaps I did not state this clear enough.
In bacteria there is no such thing as VDJ recombination.
And I dont like to link systems we use in bacteria/yeast for human or animal systems/genes.

Also: epPCR can indeed be used as a tool to study what happens with bacteria/yeast for example and you can indeed call it fast evolution, but it doesnt really represent 100% what happens in nature.
Its just a tool to cause mutation, nothing more.
ANd yes, you could state that those mutation (or some) would indeed also happen in nature , but nature is far more complex.

Also: linking genome shuffeling and epPCR is a bridge to far for me.
epPCR is much more controlled while genome shuffeling is (or can be) less controlled and you can get stranger results.
Altough, in the end its all about how you define certain stuff.

Also, and you said it yourself: its called "directed" evolution, thats just it: we (the researchers) direct evolution.. you cant simple say: aha, this is what would happen in nature.
Its a bit easy to state that.

What you create in the lab, I wouldnt call it evolution. I would call it: an observation of changes in DNA that cause a certain (observed/measured) effect, which in the end could indeed be a representation of a certain (possible) evolution.
You need to keep in mind that many of the so called "evolutions" caused by direct evolution techniques would not survive in nature or stay evolved like this because we want this evolution and keep in, while in nature the evolution might we "stupid" and not wanted and thus be lost in the end.

But this is more about semantics.

But do not agree with "directed evolution is defined as fast-forwarding natural evolution in a test tube" because for me this is not correct. A lot of the so called "direct evolution" are no evolutions that would happen in nature.


thanks.
@ pito : as you said ''epPCR can indeed be used as a tool to study what happens with bacteria/yeast for example and you can indeed call it fast evolution, but it doesnt really represent 100% what happens in nature..'' i agree with you as directed evolution could not represent 100% of nature. But it do represent some probabilistic value for evolution if we do want to believe in transition, transversion, recombination as means of evolution.



Yes,it does represents some possibilities.
Thats the idea behind it, but you should never forget what you are really doing compared with evolution

-pito-

prabhubct on Thu Aug 23 05:52:13 2012 said:




ascacioc on Wed Aug 22 19:06:14 2012 said:


@Pito: genome shuffling happens in nature: V(D)J recombination is DNA shuffling; this is the way your body makes antibodies by combining the alleles in the MHC class I loci; moreover in the next step, your body evolves the produced antibodies by epPCR-like process; both methods used by directed evolution happening in your body 4 days after infection with a virus.

The point of epPCR is that it actually recreates the same most probable mutations/errors done by DNA polymerases in bacteria. There are tons of papers calculating the probabilities of certain transitions/transversions and the hot spots for mutations and they are pretty much the same no matter what polymerase you are using. So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube. (some people use this cliche over and over again in the directed evolution field)

@prabhubct: if you would like to read more about directed evolution: http://www.sesam-bio...ected-evolution A pretty good review of the state of the art 2-3 years ago (I wrote it:P) It also has references to material that paraallels directed evolution to natural evolution.

Andreea



@ asacioc as you said '' So what you get in the test tube is what nature produces in millions of years. This is why directed evolution is defined as fast-forwarding natural evolution in a test tube.'' could it be fast- back-warding natural evolution?


You just cause mutations, backwarding or forwarding.. who knows...

Its a bit more complicated. But we are speaking of bacteria here, so there is no real telling if we are going forward of backwards... try to define backwards and forwards.. is not that easy in certain circumstances.

-pito-

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

-ascacioc-

ascacioc on Thu Aug 23 21:43:09 2012 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


you are right about, my mistake.

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 misread your first post and I was talking more about what went on before the mutation.

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:

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:

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.

All you can do is mimick what you think happens in nature, you define those boundaries.. A lot of "you" here and not a lot of "nature".

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:

@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

You only know how to improve stuff.. not to make them worse.. well there you go... evolution doesnt work like that at all.
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.

-pito-

I was not talking about not agreeing with Immunology; I was talking about the directed evolution field. I used to work (3 years) in directed evolution. I only took a specialization class in Immunology (still, I can say that I have a bit more knowledge than the average trained biologist in immunology). So... the directed evolution people really think that they are simulating evolution in their test tubes. BTW, FYI the view of one of these people about directed evolution:
http://www.chymiatrie.de/index.php/component/content/article/133-video-37

One of the popes in directed evolution explaining some things here.

And now, let's stop the discussion and call it a truce because we are getting too philosophic, polemic and semantic and too little scientific :)

Andreea

-ascacioc-
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