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1、 GoldenGateShuffling:AOne-PotDNAShufflingMethodBasedonTypeIIsRestrictionEnzymesCarolaEngler.,RamonaGruetzner.,RomyKandzia,SylvestreMarillonnet*IconGeneticsGmbH,BiozentrumHalle,Halle,GermanyAbstractWehavedevelopedaprotocoltoassembleinonestepandonetubeatleastnineseparateDNAfragmentstogetherintoanaccep
2、torvector,with90%ofrecombinantclonesobtainedcontainingthedesiredconstruct.Thisprotocolisbasedontheuseoftype IIsrestriction enzymes andis performedbysimplysubjectinga mix of10undigested inputplasmids (nineinsertplasmidsandtheacceptorvector)toarestriction-ligationandtransformingtheresultingmixincompet
3、entcells.Theefficiencyofthisprotocolallowsgeneratinglibrariesofrecombinantgenesbycombininginonereactionseveralfragmentsetspreparedfrom different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from threeparentaltemplates(bovinecationictrypsinogen,bovinea
4、nionictrypsinogenandhumancationictrypsinogen)eachdividedin9separatemodules.Weshowthatoneroundofshufflingusingthe27trypsinogenentryplasmidscaneasilyproducethe19,683differentpossiblecombinationsinonesinglerestriction-ligationandthatexpressionscreeningofasubsetofthelibraryallowsidentificationofvariants
5、thatcanleadtohigherexpressionlevelsoftrypsinactivity.Thisprotocol,thatwecallGoldenGateshuffling,isrobust,simpleandefficient,canbeperformedwithtemplatesthathavenohomology,andcanbecombinedwithothershufflingprotocolsinordertointroduceanyvariationinanypartofagivengene.Citation: Engler C, Gruetzner R, Ka
6、ndzia R, Marillonnet S (2009) Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs RestrictionEnzymes.PLoSONE4(5):e5553.doi:10.1371/journal.pone.0005553Editor:JeanPeccoud,VirginiaTech,UnitedStatesofAmericaReceivedFebruary3,2009;AcceptedApril19,2009;PublishedMay14,2009Copyright: 20
7、09 Engler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.Funding:TheworkhasbeenfundedbyIconGeneticsGmbH,Germany.Althought
8、heresearchhasbeenperformedinaprivatecompany,thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublishorpreparationofthemanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:marillonneticongenetics.de.Theseauthorscontributedequallytothiswork.Intr
9、oductionsuccessive steps, and in many cases still rely on PCR foramplification of the library since only small amount of recombi-nanttemplatesisobtained.Current protocols for assembling variant gene libraries haveevolved from the relatively simple early protocols that generatedrandom variability thr
10、ough error prone PCR 1 into a richvarietyofprotocolsthatallowsintroductionofvirtuallyanytypeofvariationinanygivengene2,3,4.Forexample,librariescanbeconstructed from pools of DNaseI digested fragments preparedfrom parental templates 5,6,7,8, from degenerate oligonucleo-tides 9,10 or from mixtures of
11、both, or even from undigestedparentaltemplates11,12,13,andareusuallyassembledthroughPCR. Libraries can also be made from parental sequencesrecombined in vivo or in vitro by either homologous or non-homologousrecombination14,15,16.WehaverecentlydevelopedaprotocolthatallowssubcloningaDNA fragment from
12、 one plasmid to another with very highefficiencyinonetubeandonestep21.Thisprotocolisalsobasedon the use of type IIs restriction enzymes, and allows theconversionofmorethanhalfofallinputplasmidsintothedesiredrecombinant product in just a 30minutes restriction-ligation.High efficiencywasalso reportedf
13、or thecloningofoneto threePCRproductsusingasimilarcloningstrategy22.Wehavenowdevelopedaprotocolforcloningmultiplefragmentsatonce,andshow here that at least 9 different fragments can be assembledtogether in a defined linear order and inserted into a recipientplasmidinonestep,andthatsuchprocedureissoe
14、fficientthatthemajority (about 90%) of colonies growing on selection platescontain the desired constructs. This efficiency is sufficient togenerate libraries of recombinant genes from several parentaltemplates.DespitethelargediversityofexistingDNAshufflingprotocols,standard cloning methods based on
15、restriction enzymes are notwidelyusedintheseprotocols.Oneobviousreasonisthatcurrentcloning methods are usually not efficient enough to generate thelarge number of variants required for DNA shuffling. Usingrestriction enzymes would have several advantages such asproviding the ability to shuffle genes
16、 irrespective of their degreeof homology, providing flexibility and control regarding thenumber of recombination events in each shuffled gene, and theability to shuffle very large genes or several regions within largegenes(independencefromPCRamplification).Infact,twoDNAshufflingstrategieshaveearlier
17、beendevelopedbasedontheuseoftype IIB or type IIs restriction enzymes 17,18,19,20. However,these protocols are quite complex to perform, require severalWe have used trypsinogen as a test protein for this shufflingprotocol. In plants, trypsinogen (bovine cationic trypsinogen) isexpressed onlyat alowle
18、vel(23,and ourunpublished results),and we suspect that this is due to instability of the protein. Wehave shuffled trypsinogen together with the genes for bovineanionic and human cationic trypsinogen. Screening of just 225recombinantclonesbytransientexpressioninNicotianabenthamianaleavesledtoselectio
19、nofvariantsthatallowsproductionofahigheramountoftrypsinactivitypergramofleaftissue.PLoSONE | www.plosone.org1May2009 | Volume 4 | Issue 5 | e5553 One-PotDNAShufflingMethodResultsDNAshufflingstrategyIn an earlier work, we have shown that a DNA fragment ofinterestcanbesubclonedwithveryhighefficiencyin
20、onestepandonetubefromoneplasmidtoanother21.Theprincipleofthecloning strategy is based on the ability of type IIs restrictionenzymes to cut outside of their recognition site. Two DNA endscanbedesignedtobeflankedbyatypeIIsrestrictionsitesuchthatdigestion of the fragments removes the enzyme recognition
21、 sitesandgeneratesendswithcomplementary4ntoverhangs;suchendscanbeligatedseamlessly,creatingajunctionthatlackstheoriginalsite(Fig.1A).Thispropertyallowscloningtobeperformedusingaone-step restriction-ligation. This strategy was shown to result intheconversionofmorethanhalfofallinputplasmidspresentinto
22、thedesiredrecombinantproductinjusta30minutesrestriction-ligation.Subcloningwasalsofoundtobeveryefficientwhentwoand three inserts were subcloned, but the total amount ofrecombinedplasmidwaslower.ThiscloningstrategycouldalsobeusedforDNAshufflingiftheentry modules that are subjected to restriction-liga
23、tion areprepared from a set of homologous genes rather than from asinglegene.SuchaDNAshufflingprotocolwouldconsistoffirstselectinganumberof4nucleotidesrecombinationsites(sequencef1tofn+1,Fig.1B)onanucleotidesequencealignmentofseveralhomologous genes. Recombination sites would be chosen onsequences t
24、hat are identical among all homologues, but differentfromallotherselectedsiteswithinthesamegene.Theselectionofthese recombination sites defines modules that consist of a coresequence (C, sequence variable among homologues) flanked bytwo 4nt sequences (f). These modules can be amplified by PCRwithpri
25、mersdesignedtoaddflankingBsaIsitesoneachsideofthemodules (the BsaI cleavage sites perfectly overlapping with therecombinationsites),andclonedinanintermediatecloningvectorand sequenced. A restriction-ligation performed on a mixcontainingallintermediateplasmids(totalnumberofplasmids:xmultipled by n),
26、the recipient acceptor vector, BsaI enzyme andligaseisexpectedtoallowassemblyofalibraryofshuffledgenes.Thisisbecauseeachmoduleiscompatibleandcanbeligatedonlytoamodulebelongingtothenextconsecutivesetofhomologousmodules,ortotheacceptorvectorforthefirstandlastmodules,andbecauseeachmodulefromasetofhomol
27、ogousmodulescanbeligatedwithequalprobabilitytoeachmoduleofacontiguousset.Inaddition,becauseoftherestriction-ligation,onlythedesiredassembled products are expected to accumulate since all otherligationproducts(forexample,ligatedproductscontainingplasmidbackboneDNAfromtheintermediateconstructs)willcon
28、tainBsaIsitesandshouldthereforebeimmediatelyredigestedwithBsaI.Asafirststeptowardtestingthisprotocol,wedecidedtotrytoassemble a plasmid from 10 separate input plasmids (9 moduleplasmidsandonevectorplasmid)inarestriction-ligation.Figure1.DNAshufflingstrategy.(A)TwoDNAendsterminatedbythe same 4 nucleo
29、tides (sequence f, composed of nucleotides 1234,complementary nucleotides noted in italics) flanked by a BsaIrecognition sequence, B, form two complementary DNA overhangsafterdigestionwithBsaI.(B)Forshuffling,genesofinterestarealigned,andrecombinationpointsconsistingof4 nucleotidesequences(f1tofn+1)
30、 are defined on conserved sequences. Module fragments (coresequence,C1toCn,plusflanking4nucleotidesequences)areamplifiedbyPCRandclonedinanintermediatecloningvector.Modulefragmentplasmids and the acceptor vector are assembled in one restriction-ligation with BsaI and ligase. S1 and S2, two different
31、selectablemarkers.Z,lacZalphagenefragment.One-potone-stepassemblyofaGFPconstructfrom10constructsWe chose to make a construct containing a GFP gene with 4introns and 5 exons (the same construct as described previously21 but with introns). The introns and exons were defined asseparate modules (sequenc
32、e of the flanking BsaI restriction sitesshowninFig.2,sequenceofthecompletemodulesgiveninFig.S1).The9fragmentswereamplifiedfromaclonedGFPgene(fortheGFPexons)orfromArabidopsisthalianagenomicDNA(fortheintrons),andclonedintotheSmaIsiteofpUC19spec(aderivativedoi:10.1371/journal.pone.0005553.g001The recip
33、ient expression vector, pX-LacZ (Fig. 2, describedpreviouslyin21)containstwoBsaIsitescompatiblewiththefirstandlastexonmodules.of pUC19 with the ampicillin-resistance b-lactamase genereplaced by a spectinomycin-resistance gene) and sequenced.PLoSONE | www.plosone.org2May2009 | Volume 4 | Issue 5 | e5
34、553 One-PotDNAShufflingMethodFigure 2. Assembly of a GFP construct from 10 plasmids. (A) Construct maps. Input modules contain a core region C flanked by BsaIrestrictionsitesinoppositeorientationcomposedofarecognitionsite(B,ggtctcn,B,ngagacc)anda4nucleotidecleavagesite(boxesflankingthecore region).
35、pX-LacZ, acceptor vector. pGFPi, resulting construct. Restriction sites for AvrII and XmaI are shown as white arrows. (B ) Ethidiumbromide-stained gel with products obtained by restriction-ligation of the 9 input module plasmids. M: GeneRuler 1kb DNA Ladder Plus fromFermentas.Restriction-ligationwas
36、performedat37uCfor3(lane3h)or6hours(lane6h)orwith25cycles(2min37uC+5min16uC,lane25)or50cycles (lane50), and withoutBsaI enzyme(lanenb). Thearrowindicatesthe 1.17kblinear assembled GFP geneproduct. (C) Ethidiumbromide-stained gels of 72 minipreps digestedwithXmaI and AvrII(expected fragmentsizes: 4.6
37、kb, 945 and 555bp), obtainedfrom restriction-ligationsperformedfor6h37uC(6h),for25or50cycles(256/506),withnormalligase(nl)orhighconcentrationligase(hcl).Numbersindicateminiprepswithanincorrectrestrictionpattern,andstarsindicateconstructsthatconsistofdimers(samerestrictionpatternasmonomers).V,vectorp
38、X-lacZ.doi:10.1371/journal.pone.0005553.g002Todefineoptimalrestriction-ligationconditions,afirstwas visible on agel even when normal ligase wasused, and washighestafter50cyclesexperimentwasperformedusingonlythenineGFPintron/exonconstructs without the acceptor vector. The result of therestriction-lig
39、ation is expected to be a 1.17kb linear fragmentcontainingtheassembledGFPexonsandintrons,inadditiontoalllinear entry vector backbone fragments (2.8kb). Restriction-ligationsweresetupbypipettingintoatube75ngofeachofthe9plasmids(5exons,4introns),2.5unitsofBsaIenzyme(NEB)andeither 2.25 or 15 units of T
40、4 DNA ligase (Promega, 0.75m l ofnormal -3u/ml or high concentration HC ligase - 20u/ml,respectively) in atotalvolume of15microliters inligation buffer(Promega).Therestriction-ligationswereincubatedat37uCfor3and6hoursandthenrunonanagarosegel.Theexpected1.17kbbandcouldbeseenonlywhentheligationwasperf
41、ormedwithHCligase, and mostly after a 6hour ligation (Fig. 2B). To try toimprove the amount of assembled ligated product, we modifiedthe restriction-ligation parameters so as to alternate betweenconditionsoptimalforannealingoftheDNAendsandconditionsoptimalforenzymaticreactions(digestionorligation),a
42、ndforthispurpose, performed the restriction-ligation in a thermocycler.Programs were defined with the following steps: incubation for2minutes at 37uC and 5minutes at 16uC, both steps repeatedeither 25 or 50 times, followed by incubation for 5minutes at50uC (final digestion) and then 5minutes at 80uC
43、 (heatinactivation). These conditions were more efficient than acontinuous incubation at 37uC because the expected productThesameconditionsasdescribedabovewerealsousedwithamix containing the acceptor expression vector in addition to thenineGFPmoduleplasmids(75ngofeachofthe10plasmids).Theligation was
44、 transformed into 100microliters of chemicallycompetent DH10B cellsand 20mlout ofa final volume of1mlplated on Kanamycin X-gal plates. For all restriction-ligationsperformed with BsaI and ligase, the number of white coloniesmirrored the efficiency of ligation observed in the ligation assaydescribed
45、above (Table 1). In general, high concentration ligasewas more efficient than normal ligase for restriction-ligationsperformedwithoutcycling,butbothnormalandhighconcentra-tionligasesappearedtoworkwellwithaprogramwith50cycles.PlasmidDNAwaspreparedfrom12whitecoloniesforeachof6transformations (6hr 37uC
46、, 25 cycles and 50 cycles, each withnormal and HC ligase) and was analyzed by gel electrophoresisundigested or digested with XmaI and AvrII. Analysis ofundigested DNA (not shown) indicates that 4 out of 72 clonesconsisted of dimers (vector-insert-vector-insert religated, star inFig. 2C). Analysis of
47、 digested DNA revealed that 67 out of 72clones had the expected restriction pattern, or 93% of whitecolonies.Whenbothincorrectinsertsanddimersareincluded,thisleadstoasuccessrateof63correctcoloniesoutof72,or87.5%ofallwhitecolonies.Byextrapolatingthisfrequencyofcorrectclonesto the entire transformation, one can conclude that up to 7918PLoSONE | www.plosone.org3May2009 | Volume 4 | Issue 5 | e5553 One-PotDNAShufflingMethodTable1.AssemblyofpGFPifrom10plasmids.Ligationconditionsneg3h37uC6h37uC25cycles50cycles20m lplatedblue/white20m lplatedblue/whiteestimatedtotalcorrect20m lplatedblue/whitees