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36 replies to this topic

#31 Aegist

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Posted 12 January 2007 - 04:33 AM

[spectate]

#32 Ghostrider

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Posted 12 January 2007 - 05:18 AM

Why distorted? Is this because you don't understand the principles of phage display it or because you refuse to acknowledge that a fundamentally important methodology has been overlooked?  It seems an unfortunate personality trait that when you cannot defend your position you must resort to ad hominem attacks.


Prometheus, John already gave his reason for why he favors RNA libraries over phage display - efficiency:

"However, I favor RNA libraries over phage display for reasons outlined above (such as seven orders of magnitude more sequences that can be accessed per experiment)."

Is he overlooking something? What specifically is the flaw in his reasoning that motivates you to object so strongly? By the way, I don't think John made any ad hominem attacks in this thread.

John, I never heard a direct rejection for Prometheus's suggestion on the previous page:

In any case, as I have pointed out above, turnover is but one way of
characterizing an enzyme. Binding specificity to its substrate is
another.

But you raise an important point, and that is the development of an
assay that can discriminate between binding affinity versus chemical
reactivity from the results of a phage display methodology to generate
fucntional diversity. For example, to take the results of phage
mediated evolution and then link to a mechanism that can report
production of product. In the studies performed in our lab we use
streptavidin magnetic beads which can bind to biotinylated cells for
cell sorting. One could presumably - I don't know if this has been
done before - use a similar mechanism to separate small groups of
phage (by biotinylating them) and then monitoring for turnover in
individual wells.


Is the method that he is suggesting simply less efficient than the RNA library approach? What makes his suggestion less efficient or inapplicable?

I would be very suprised if you were to have your supervisor agree that rummaging around graveyards in the hope of identifying a novel enzyme presents a superior methodological approach to directed evolution.


Prometheus, I see no harm in searching for a needle in a haystack if one knows that is where the needle truly lies. Certainly, the most efficient method for searching must be used as lives are being lost each day. You indicated above that phage display is the most efficient method for this enzyme search. John gave his reasons for why he believes that RNA libraries are the best way to find our enzyme of interest. Please use this opportunity to either show what John has overlooked or provide proof that phage display is a better approach than RNA libraries for the task at hand.

#33 Karomesis

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Posted 12 January 2007 - 02:28 PM

forgive my ignorance gentlemen, but is there a site or paper with some drawings of these processes on them?

I find it easier to learn in unorthodox ways, especially about stuff like this. [glasses]

#34 John Schloendorn

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Posted 13 January 2007 - 01:19 AM

John, I never heard a direct rejection for Prometheus's suggestion on the previous page.

That's because it was no suggestion at all, but a collection of gaps. Just how do you want me to "take the results of phage mediated evolution and then link to a mechanism that can report production of product"? Some people have been very clever about how to do this for specific purposes under specific contraints, but a generally applicable method do do this is just inexistent. I neither know what the ideal product would be, nor an assay for it, no high throughput screening capability to "monitor individual wells", no enzyme to begin with, no information whether such an enzyme or its mutants would be active as a phage fusion, no possibility to do it on more than one enzyme at a time, no personell to actually do any of this, and results from grave-derived bacteria that already suggest here and now that none of this would be necessary even if it were possible. And now explain to me why you don't use a lawnmower to brush your teeth please.

Even the far more powerful mRNA display offers no convincing solution to do what I already successfully did within one person year with grave-derived microbes. But I believe it may have applications for other problems, especially when the target is not a pathway, but a single enzyme.

#35

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Posted 13 January 2007 - 07:57 AM

Tiddlywinks.

#36 enki273

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Posted 13 January 2007 - 12:24 PM

excuse my ignorance, but what are tiddlywinks?

#37 Ghostrider

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Posted 13 January 2007 - 11:12 PM

Tiddlywinks.


Prometheus, it seems that you disagree with what John has stated. Please use this time as an opportunity to bolster your point of view by describing what John has overlooked or misunderstood. This would be a good place to start:

Just how do you want me to "take the results of phage mediated evolution and then link to a mechanism that can report production of product"? Some people have been very clever about how to do this for specific purposes under specific contraints, but a generally applicable method do do this is just inexistent. I neither know what the ideal product would be, nor an assay for it, no high throughput screening capability to "monitor individual wells", no enzyme to begin with, no information whether such an enzyme or its mutants would be active as a phage fusion, no possibility to do it on more than one enzyme at a time, no personell to actually do any of this, and results from grave-derived bacteria that already suggest here and now that none of this would be necessary even if it were possible.






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