in vitro compartmentalization

Mix up genomic library with cell-free expression system and oil.
End up with oil droplets with DNA plus expression system.

Advantages? Not sure

Apparently better than other in vitro selection techniques such as phage display/ribosome display/mRNA peptide fusion/SELEX (would like to read about these too)

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Paper on DNA shuffling of Fe-Fe hydrogenases
-> heterologous expression of h2ase in E coli:D
Looks to be similarly possible for Ni-Fe.

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Directed evolution of oxygenases:
Table 1 compares selection methods

FACS pretty good (10^7). Phage display probs wouldn't work cos the protein wouldn't fold properly. Solid media a bit iffy, but I think it would work well if there was a selection pressure on the cells.

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Enzyme (re)design: lessons from natural evolution and
computation
John A Gerlt1 and Patricia C Babbitt

"Aggressive enzyme engineering". Instead of making small changes, tear off massive chunks of the protein and add on new loops.

Use computers (RosettaMatch) to work out what loops will work well on what scaffold.
Follow it with directed evolution to optimise function.

"Enzyme (re)design: lessons from natural evolution and
computation"
- functionally diverse superfamilies: same catalytic method, different reactions
- can create new catalysts by taking an original enzyme and modifying its specificity (same catalytic method)
- usually occurs at expense of original functionality
- other option: some enzymes are promiscuous: use directed evolution to favour one reaction.

"Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation"

Ile-insensitive mutant designated ilvA-466 + l-O-methylthreonine (OMT) = death?

wild-type ilvA -> life?