Biomedical Engineering Reference
In-Depth Information
(a)
(b)
Figure 3.2
(See companion CD for color igure.) .Directed.evolution.and.PACE.schematics..(a).Traditional.directed.evolution.
consists.of.discrete.rounds.of.sequential.mutagenesis.and.selection..Mutagenesis.of.a.parent.gene.(left).generates.
a. library. (top). with. diverse. mutations. (red. x's).. The. mutant. genes. are. transformed. into. bacteria. (right),. which.
transcribe.and.translate.the.genes.into.mutant.proteins.(colored.circles)..A.screen.or.selection.identiies.the.ittest.
mutants,.the.gene.is.isolated.and.ampliied,.and.another.cycle.begins..(b).PACE.automates.the.process,.allowing.
dozens.or.hundreds.of.rounds.to.proceed.without.human.intervention..Bacteria.(tan.ovals).low.into.a.“lagoon”.
(black-bordered.rectangle).containing.phage.(blue.rectangles)..The.phage.infects.the.bacteria,.which.express.the.
target.gene.contained.within.the.phage.genome.(black.ovals.with.red.x's.indicating.mutations),.producing.mutant.
protein.(colored.circles)..Only.mutant.proteins.with.the.desired.activity.induce.expression.of.the.pIII.protein.(blue.
stars).from.a.plasmid.in.the.bacteria.(green.ovals)..Newly.produced.phage.without.pIII.are.not.infectious..A.gene.
must.produce.copies.of.itself.within.infectious.phage.rapidly.enough.to.avoid.washing.out.of.the.lagoon.
function.optimally.within.nonnatural.environments.or.on.nonnatural.substrates,.protein.
engineers.have.mimicked.the.evolutionary.process.by.which.these.enzymes.were.created..
Directed. evolution. is. a. laboratory-based. method. in. which. humans. replace. the. natural.
selective. pressures. that. determine. the. itness. of. a. protein. with. nonnatural,. application-
speciic. selective. pressures.. A. diverse. collection. (called. a. “library”). of. mutants. based.
on. a. natural. parent. protein. is. generated,. and. each. candidate. is. tested. for. the. desired.
function..The.candidates.deemed.the.ittest.under.the.conditions.of.the.inal.application.
are.replicated.and.mutated,.providing.mutants.for.a.new.round.of.directed.evolution..This.
cycle.is.repeated.until.a.suitable.enzyme.is.found.(Figure.3.2a).
Each. directed. evolution. project. requires. a. customized. high-throughput. selection.
or. screen. capable. of. identifying. improved. enzymes.. In. vivo. selections. and. screens. are.
convenient.and.popular.but.are.limited.by.the.eficiency.of.transforming.mutant.DNA.into.
the.host.and.by.the.tendency.of.cells.to.ind.alternative,.undesired.ways.of.avoiding.the.
selection.pressure..Extremely.high.throughput.in.vitro.techniques.such.as.ribosome.display.
(Hanes.and.Pluckthun.1997).partially.address.these.limitations,.but.have.been.restricted.
to.selections.for.binding..Recently,.however,.methods.including.yeast.display.(Chen.et.al..
2011),.mRNA.display.(Seelig.and.Szostak.2007),.and.in.vitro.compartmentalization.(IVC;.
Tawik.and.Grifiths.1998).have.been.adapted.for.the.selection.of.active.enzymes..IVC,.in.
particular,.is.extremely.promising.as.it.can.select.for.a.wide.variety.of.multiple-turnover.
enzymatic.activities.(Stapleton.and.Swartz.2010).
Recently,.a.phage-based.technique.has.been.developed.that.allows.continuous.directed.
evolution.of.proteins.(Esvelt.et.al..2011),.as.opposed.to.the.discrete,.labor-intensive.rounds.
of. traditional. methods. (Figure. 3.2b).. Phage-assisted. continuous. evolution. (PACE). takes.
advantage. of. the. rapid. life. cycle. and. high. mutation. rate. of. Escherichia coli -infecting.
bacteriophage..Fresh. E. coli .cells.low.into.a.well-mixed.volume.termed.a.“lagoon,”.where.
they.remain.for.a.residence.time.shorter.than.their.division.period.before.lowing.to.waste..
Within.the.lagoon.is.a.population.of.phage.that.is.diluted.by.the.continuous.low.and.must.
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