Talk:CH391L/S12/In vitro Selection

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Concerning that in vitro compartmentalisation, if say, applied for measuring enzymatic functions, would that oil compound affect the activity of the enzyme? how would you control the inside temperature, and would that temperature change cause a change in the size of the compartment(since CMC depends on temp)?*Yi Kou 13:04, 31 January 2012 (EST):

  • Adam Meyer 17:35, 9 February 2012 (EST):Yes, the oil and/or surfactant can interfere with protein function in a emulsion. The temperature is the same as ambient temperature. I know little about the link between radius and temperature, but emulsion PCR (involving many temperature changes) is common.

How is it that you can isolate specific protein or gene products in oil bubbles in an emulsion of oil and water? 16:53, 31 January 2012 (EST)

  • Adam Meyer 17:35, 9 February 2012 (EST):There are more compartments than there are DNA templates, so the majority of compartments will have zero or one template. The gene product is made within the compartments and thus is confind to the same bubble as its template. Multiple templates per compartments is a problem that can allow an inactive species to survive a round of selection. This is one of the reasons that multiple rounds are usually necessary.

  • Jeffrey E. Barrick 11:32, 5 February 2012 (EST):Overall organization is good. It would be nice to have a better introduction at the beginning of this topic. It jumps right into details without explaining where it's going.

  • Jeffrey E. Barrick 11:32, 5 February 2012 (EST):I think all the schemes are "Selective Amplification" and that the ribozyme scheme might be better called "Self-modification"?
  • Adam Meyer 17:35, 9 February 2012 (EST):I differentiate between amplification in the selection phase (like when selecting a polymerase) and amplification after , the selection (like when selecting for aptamer, antibody, ligase, etc.)

  • Jeffrey E. Barrick 11:32, 5 February 2012 (EST):To flesh out some important but neglected sections, you should really add at least one directed evolution reference (for a plasmid library selected in cells for a function). Maybe a Frances Arnold study where they change enzyme substrate specificity or thermostability?
  • Adam Meyer 17:35, 9 February 2012 (EST):I thought that there was already too much and nobody would want to hear me talk more than I did.

  • Erik Quandt 22:50, 5 February 2012 (EST): I thought that phage display deserved a mention
    • Adam Meyer 17:35, 9 February 2012 (EST):I thought that selecting for protein-binding (including phage display, ribosome display, cell-surface display, and various emulsion-based systems) was too big a topic to cover in addition to what I did. Although it is arguably the most successful facet of in vitro selection.
      • Jeffrey E. Barrick 12:02, 11 February 2012 (EST):It seems like someone needs to cover phage display and directed protein evolution, but I agree that this topic is too big already!

  • David M. Truong 23:03, 5 February 2012 (EST): I'm curious about what you do when your in vitro selected product doesn't work in vivo (or at least not as well)? How do the limitations of in vitro selection go hand-in-hand with in vivo selection experiments, and vice versa?
  • Adam Meyer 17:35, 9 February 2012 (EST):It is common for selected variants to be optimized to function in the selection (and not where you actually want to use it). The best thing you can do is design your selection scheme to closely mimic the conditions in which the final product will be used. You get what you select for.
  • Jeffrey E. Barrick 10:40, 13 February 2012 (EST):I think there's been a recent trend (at least for allosteric ribozymes and riboswitches) to select at lower Mg2+ concentrations to achieve better in vivo activity. I'm not familiar with any selections in cell lysate -- which seems like it would be the best way to achieve in vivo function. Maybe that is too challenging, and RNAs degrade too rapidly.