Welcome to Scribd. Sign in or start your free trial to enjoy unlimited e-books, audiobooks & documents.Find out more
Standard view
Full view
of .
Look up keyword
Like this
0 of .
Results for:
No results containing your search query
P. 1
Nature 09929

Nature 09929

|Views: 49|Likes:
Published by Nathan McCorkle

More info:

Published by: Nathan McCorkle on Apr 21, 2011
Copyright:Attribution Non-commercial


Read on Scribd mobile: iPhone, iPad and Android.
download as PDF, TXT or read online from Scribd
See more
See less





A system for the continuous directed evolution of biomolecules
Kevin M. Esvelt
, Jacob C. Carlson
& David R. Liu
Laboratory evolution has generated many biomolecules withdesiredproperties,butasingleroundofmutation,geneexpression,screening or selection, and replication typically requires days or longer with frequent human intervention
. Because evolutionary success is dependent on the total number of rounds performed
, a means of performing laboratory evolution continuously andrapidly could dramatically enhance its effectiveness
. Althoughresearchers have accelerated individual steps in the evolutionary cycle
,theonlypreviousexampleofcontinuousdirectedevolutionwas the landmark study of Wright and Joyce
, who continuously evolvedRNAligaseribozymeswithan
in vitro 
Escherichia coli 
. During phage-assisted continuous evolution(PACE), evolving genes are transferred from host cell to host cellthrough a modified bacteriophage life cycle in a manner that isdependentontheactivityofinterest.Dozensofroundsofevolutioncan occur in a single day of PACE without human intervention.UsingPACE,weevolvedT7RNApolymerase(RNAP)variantsthatrecognizeadistinctpromoter,initiatetranscriptswithATPinsteadof GTP, and initiate transcripts with CTP. In one example, PACEexecuted 200 rounds of protein evolution over the course of 8days.Starting from undetectable activity levels in two of these cases,enzymeswitheachofthethreetargetactivitiesemergedinlessthan1week of PACE. In all three cases, PACE-evolved polymeraseactivities exceeded or were comparable to that of the wild-type T7RNAPonitswild-typepromoter,representingimprovementsofupto several hundred-fold. By greatly accelerating laboratory evolu-tion,PACEmayprovidesolutionstootherwiseintractabledirectedevolution problems and address novel questions about molecular evolution.
We devised a system that exploits the continuous culture and selec-tionoftheM13filamentousbacteriophage
) to enable the continuous directed evolution of proteins ornucleic acids. In PACE,
E. coli
host cells continuously flow through afixed-volume vessel (the ‘lagoon’) containing a replicating populationof phage DNA vectors (‘selection phage’) encoding the gene(s) of interest (Supplementary Fig. 1).Theaverageresidencetimeofhostcellsinthelagoonislessthanthetime required for
E. coli
replication. As a result, mutations accumulateonlyintheevolvingselectionphagepopulation,theonlyDNAthatcanreplicate faster than the rate of lagoon dilution. The mutation of hostcells in the lagoon should therefore have minimal impact on the out-come of the selection over many rounds of phage replication, andmutagenesis conditions are not limited to those that preserve
E. coli
 viability.PACE achieves continuous selection by linking the desired activity to the productionof infectious progeny phage containing the evolving gene(s).PhageinfectionrequiresproteinIII(pIII;encodedbygeneIII),whichmediatesFpilusbindingandhostcellentr
.PhagelackingpIIIare approximately 10
-fold less infectious than wild-type phage
.Crucially, the production of infectious phage scales with increasing levels of pIII over concentrations spanning two ordersof magnitude
.TocouplepIIIproductiontotheactivityofinterest,wedeletedgeneIII from the phage vector and inserted it into an ‘accessory plasmid’present in the
E. coli
host cells (see Supplementary Fig. 2 for plasmidmaps). The production of pIII from the accessory plasmid is depend-enton the activity of the evolving gene(s) on the selection phage. OnlphagevectorsabletoinducesufficientpIIIproductionfromtheaccess-ory plasmid will propagate and persist in the lagoon (Fig. 1). BecausepIII expression level determines the rate of infectious phage produc-tion
, phage encoding genes that result in a higher level of pIII pro-duction will infect more host cells than phage encoding less activegenes.Owing to the speed of the phage life cycle (progeny phage produc-tionbeginsapproximately10minafterinfection)
,PACEcanmediatemany generations of selective phage replication in a single day. Weobserved activity-dependent phage vectors that tolerate lagoon flow rates up to 3.2 volumes per hour (Supplementary Fig. 3), correspond-ingtoanaverageof38phagegenerationsper24h(seetheSupplemen-tary Information for an analysis). More conservative flow rates of 2.0–2.5 volumes per hour allow 24–30 generations per day and reducethe risk of complete phage loss (washout) during selections. Multiplelagoons can evolve genes in parallel, with each 100ml lagoon contain-ing approximately 5
host cells selectively replicating activephage variants. Importantly, PACE requires no intervention during evolution and obviates the need to create DNA libraries, transformcells, extract genes or perform DNA cloning steps during each round.Inprinciple, PACEiscapableofevolvinganygenethatcanbelinkedto pIIIproductionin
E. coli
. Because a widevarietyoffunctions includ-ing DNA binding, RNA binding, protein binding, bond-forming cata-lysis and a variety of enzyme activities have been linked to the expres-sion of a reporter protein
, PACE can be applied to the evolution of manydifferentactivitiesofinterest.Asexamples,wesuccessfullylinkedprotein–protein binding, recombinase activity and RNAP activity tophage infectivity in discrete infection assays by creating variants of the accessory plasmid that associate each of these activities with pIIIproduction (Fig. 2).PACE applies optimal evolutionary pressure when pIII levels areabove the minimal threshold required to prevent phage washout, butbelow the amount needed to maximize infectious phage production.Thiswindowcanbeshiftedbyvaryingthecopynumberoftheaccessorplasmid, or by altering the ribosome-binding site (RBS) sequence of gene III to modulate the efficiency with which gene III is expressed(Supplementary Fig. 4).We constructed an arabinose-inducible mutagenesis plasmid thatelevates the error rate during DNA replication in the lagoon by sup-pressingproofreading 
andenhancingerror-pronelesionbypass(Sup-plementary Information)
. Full induction increased the observedmutagenesis rate by approximately 100-fold, inducing all possibletransitions and transversions (Supplementary Fig. 5). This enhanced
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge,Massachusetts 02138, USA.
Howard Hughes Medical Institute, Cambridge, Massachusetts 02138, USA.
0 0 M O N T H 2 0 1 1 | V O L 0 0 0 | N AT U R E | 1
Macmillan Publishers Limited. All rights reserved
mutation rate is sufficient to sample all possible single and doublemutants of a given phage-encoded sequence in each generation(Supplementary Information), in principle enabling single-mutationfitness valleys to be traversed during PACE.Bacteriophage T7 RNAP is widely used to transcribe RNA
in vitro
and in cells. T7 RNAP is highly specific for its promoter sequence(TAATACGACTCACTATA), and exhibits virtually no detectableactivity on the consensus promoter of the related bacteriophage T3(AATTAACCCTCACTAAA, differences underlined)
. Despitedecades of study and several attempts to engineer the specificity of T7 RNAP towards other promoters
including that of T3, a mutantT7 RNAP capable of recognizing the T3 promoter has not been previ-ously reported.To remove potential interference from evolutionary improvementstothephagevector rather thantoT7RNAP, we propagated a selectionphageexpressingwild-typeT7RNAPfor3daysonhostcellscontaining an accessory plasmid with the wild-type T7 promoter driving gIIIexpression. A single plaque presumed to represent vector-optimized
Host cells withaccessory plasmidandmutagenesis plasmid
Constant inflow
The ‘lagoon’
Constant outflow
Selection phage:encodes librarymemberSP APMPInfectionInfectiousprogenyNon-infectiousprogeny*****Mutagenesis(continuous)SP** AP*MPSP** AP*xMPIf SP inducespIII productionIf SP fails toinduce pIIIproductionFunctionallibrary memberNon-functionallibrary memberSPSP AP APxMPMP
Figure 1
OverviewofthePACE system.
PACE in a single lagoon. Host cellscontinuously flow through a lagoon, where they are infected with selectionphage (SP) encoding library members. Functional library members induceproduction of pIII from the accessory plasmid (AP) and release progeny capableofinfectingnewhostcells,whereasnon-functionallibrarymembersdonot. Increased mutagenesis is triggered through induction of the mutagenesisplasmid (MP). Host cellsflowoutof thelagoonon averagefasterthan they canreplicate, confining the accumulation of mutations to replicating phage.
SPexpressingT7 RNAPSP with noT7 RNAPWhole M13phageHost cellsonlySP withrecombinaseSP with norecombinaseGene IIIT7 promoterT7 RNAP
Polymerase activity
Protein–peptide bindingRecombinase-catalysed inversionGene IIIGene IIIPromoterPromoterRecRecRecRec
Recombinase activityLGF2Operator
Gal11pGene IIIRNAP
Zi268DNA-bindingdomainSP with noGal11p-Zi268SP withGal11p–Zi268
Figure 2
Linkage of three protein activities to pIII production and phageinfectivity using three distinct accessory plasmids.
E. coli
cells containing accessory plasmids encoding conditionally expressed gene III (left) andselection phage were combined with recipient cells. Phage production resultedin colonies with antibiotic resistances conferred by the phage and the recipientcells (right). See Methods for details.
, RNAP activity leads to gene IIIexpression and infection comparable to wild-type phage, whereas selectionphagelackingT7RNAPdonotinfect.
,Protein–proteininteractionbetweenaGal11p domain tethered to a Zif268 DNA-binding domain and an LGF2adomain fused to RNAP leads to gene III expression and infection.
, Recombinase-catalysed gene inversion induces gene III expression andinfection.
2 | N AT U R E | V O L 0 0 0 | 0 0 M O N T H 2 0 1 1
Macmillan Publishers Limited. All rights reserved
selectionphagecontainedasinglemutation(P314T)inT7RNAP.Weconfirmed that the activity of the P314T mutant does not significantly differ from that of wild-type T7 RNAP (Supplementary Fig. 6).This starting selection phage failed to propagate on host cells con-taining the T3 promoter accessory plasmid. We therefore propagatedthe phage on cells containing a hybrid T7/T3 promoter accessory plasmid with the T7 promoter base at the important
11 position
but all other positions changed to their T3 counterparts. Two initially identical lagoons were evolved in parallel on the hybrid promoteraccessoryplasmidfor60h,thenonthecompleteT3promoteraccessorplasmid for 48h, and finally on a high-stringency, very low-copy T3promoter accessory plasmid for 84h (Fig. 3a).Phagepersistedinbothlagoonsafter8daysofPACE,survivinganetdilution of 10
-fold, the equivalent of 555 phage population dou-blings and 200 rounds of evolution by the average phage. We isolated,sequenced and characterized phage vectors from each lagoon after 48,108 and 192h, observing up to 8, 10 and 11 non-silent mutations insingle T7 RNAP genes at each time point.Protein-encoding regions (without upstream promoter sequences)ofevolvedmutantT7RNAPgenesweresubclonedintoassayplasmidsthat quantitatively link transcriptional activity to
-galactosidaseexpression in cells
. We defined the activity of wild-type T7 RNAPon the T7 promoter to be 100%. The starting T7 RNAP exhibitedundetectable (
3%) levels of activity on the T3 promoter in thesecell-based assays. The assayed mutants exhibited more than 200%activity after 108h of PACE, and more than 600% activity afterhigh-stringency PACE at 192h, improvements of more than 200-fold(Fig. 3b, c). These results collectively establish the ability of PACE toevolve large changes in enzyme activity and specificity very rapidly with minimal intervention by the researcher.SeveralevolvedT7RNAPmutantswerealsopurifiedandassayed
using radioactive nucleotide incorporation assays. Purified T7RNAP mutants exhibited activity levels on the T3 promoter
in vitro
exceeding that of wild-type T7 RNAP on the T7 promoter, represent-ingimprovementsofupto89-foldcomparedwiththestartingenzyme(Supplementary Fig. 7). These results indicate that PACE resulted inlarge improvements in substrate binding or catalytic rate. Evolvedactivity improvements were higher in cells than
in vitro
, suggesting that these enzymes also evolved improvements in features such asexpression level, polymerase folding or stability that are specific tothe context of the cytoplasm.Interestingly,theevolutionarydynamicsofthetwoinitiallyidenticallagoons differed significantly (Fig. 3d and Supplementary Results).Within 24h, lagoon 1 acquired a predominant suite of mutations
T3 promoterhigh-copy APT3 promotervery low-copy AP
Time (h)024487296120
Seed lagoon withstarting T7 RNAP
Induced mutagenesisLagoon fow rate: 2.0 volumes per hour throughoutHybrid promoterhigh-copy APT3 promoterhigh-copy APT3 promotervery low-copy APHybrid promoterhigh-copy AP
Hybrid promoter
MutagenesisinducerSwitch host cells romhybrid promotertoT3 promoterduring evolution
T3 promoter
Lagoon 2
Lagoon 1T3 promotervery low-copy AP
Time (h)Time (h)Time (h)024487296120144168
T3 promoterhigh-copy APHybrid promoterhigh-copy APT3 promotervery low-copy APT3 promoterhigh-copy APHybrid promoterhigh-copy AP
   R  e   l  a   t   i  v  e   t  r  a  n  s  c  r   i  p   t   i  o  n  a   l  a  c   t   i  v   i   t  y   i  n  c  e   l   l  s   (   W   T   T   7   R   N   A   P  o  n   T   7  p  r  o  m  o   t  e  r  =   1   0   0   )
Lagoon 1
T7 promoterT3 promoter
P314T L2-48.1 L2-48.2 L2-48.3 L2-108.1 L2-108.2 L2-108.3 L2-192.1
   R  e   l  a   t   i  v  e   t  r  a  n  s  c  r   i  p   t   i  o  n  a   l  a  c   t   i  v   i   t  y   i  n  c  e   l   l  s   (   W   T   T   7   R   N   A   P  o  n   T   7  p  r  o  m  o   t  e  r  =   1   0   0   ) 
Lagoon 2
T7 promoterT3 promoter
I4MI4MI4MI4MI4MI4MI4MI4MI4MI4MI4MI4ME63VE63VE63VE63VE63VE63VK98RK98RK98RK98RN165SN165SN165SG175RG175RG175RG175RG175RG175RG175RG175RG175RG175RG175RG175RG175RG175RE222KE222KE222KE222KE222KE222KE222KE222KE222KE222KE222KQ239RQ239RQ239RQ239RQ239RQ239LQ239LP248SP248SP248SE484AE484AE484AE484AE484AE484AE484AG542VG542VG542VG542VG542VG542VG542VG542VG542VG542VG542VG542VG542V V574AV574AV574AV574AV574AV574AG675RG675RG675RG675RG675RG675RI681LI681LI681LL699IL699IN748DN748DN748DN748DN748DN748DN748DN748DE775KE775KE775KE775KE775KE775KE877KE877KE877K
024487296120144168192Lagoon 1Lagoon 2
Figure 3
ContinuousevolutionofT7RNAPvariantsthatrecognizetheT3promoter. a 
, PACE schedule.
, Activity in cells of T7 RNAP variants isolatedfrom lagoon 1 at 48, 108 and 192h on the T7 and T3 promoters.Transcriptional activity was measured spectrophotometrically by subcloning the protein-encoding regions of the T7 RNAP genes into a construct in whichthe T7 or T3 promoter drives
, Activity in cells of T7 RNAP variants isolated from lagoon 2. Error bars in
, standard deviation of atleastthreeindependentassays.
,MutationsidentifiedinT7RNAPclonesfromlagoons 1 and 2 are shown in red and blue, respectively. Underlined mutationswere predominant in that lagoon based on whole-pool sequencing of lagoonaliquots. Mutations highlighted in yellow independently evolved topredominance in both lagoons.
0 0 M O N T H 2 0 1 1 | V O L 0 0 0 | N AT U R E | 3
Macmillan Publishers Limited. All rights reserved

You're Reading a Free Preview

/*********** DO NOT ALTER ANYTHING BELOW THIS LINE ! ************/ var s_code=s.t();if(s_code)document.write(s_code)//-->