bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
Multiplexgenomeeditingbynaturaltransformation(MuGENT)
2
forsyntheticbiologyinVibrionatriegens
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4
TrianaN.Dalia1,ChelseaA.Hayes1,SergeyStolyar2,ChristopherJ.Marx2,JamesB.
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McKinlay1,andAnkurB.Dalia1,*
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1DepartmentofBiology,IndianaUniversity,Bloomington,IN47401.2Departmentof
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BiologicalSciences,UniversityofIdaho,Moscow,ID83844.
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*Authorforcorrespondence:AnkurB.Dalia,[email protected]
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12
VibrionatriegenshasrecentlyemergedasanalternativetoEscherichiacoliformolecular
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biologyandbiotechnology,butlow-efficiencygenetictoolshamperitsdevelopment.Here,
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weuncoverhowtoinducenaturalcompetenceinV.natriegensanddescribemethodsfor
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multiplexgenomeeditingbynaturaltransformation(MuGENT).MuGENTpromotes
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integrationoflargegenomeeditsathigh-efficiencyonunprecedentedtimescales.Also,this
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methodallowsforgeneratinghighlycomplexmutantpopulations,whichcanbeexploited
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formetabolicengineeringefforts.Asaproof-of-concept,weattemptedtoenhance
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productionofthevalueaddedchemicalpoly-β-hydroxybutyrate(PHB)inV.natriegensby
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targetingtheexpressionofninegenesinvolvedinPHBbiosynthesisviaMuGENT.Within1
21
week,weisolatededitedstrainsthatproduced~100timesmorePHBthantheparent
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isolateand~3.3timesmorethanarationallydesignedstrain.Thus,themethodsdescribed
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hereshouldextendtheutilityofthisspeciesfordiverseacademicandindustrial
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applications.
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V.natriegensisthefastestgrowingorganismknown,withadoublingtimeof<10min1,2.
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Withbroadmetaboliccapabilities,lackofpathogenicity,anditsrapidgrowthrate,itisan
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attractivealternativetoE.colifordiversemolecularbiologyandbiotechnology
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applications3,4.MethodsforclassicalgenetictechniqueshavebeendevelopedforV.
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natriegens,butthesearerelativelylaborious,requiremultiplesteps,andmustbeused
1
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
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1
sequentiallytogeneratemultiplegenomeedits3,4.Thechallengesofthesetechniques
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contrastwiththeeaseofgeneticsinVibriospeciesthatarenaturallytransformable.
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CompetentVibrioscantakeupDNAfromtheenvironmentandintegrateitintotheir
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genomebyhomologousrecombination;processesknownasnaturalcompetenceand
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naturaltransformation,respectively5-8.Theinducingcuefornaturaltransformationin
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competentVibriosisgrowthonthechitinousshellsofcrustaceanzooplankton,whichare
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commonlyfoundintheaquaticenvironmentwherethesemicrobesreside5.Chitininduces
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theexpressionofthecompetenceregulatorTfoX9,10.Infact,overexpressionofTfoX
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obviatestheneedforchitininduction,allowingcompetentVibriostobenaturally
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transformedinrichmedia5,9.
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AsnoreportsofnaturaltransformationexistedforV.natriegens,wefirstsoughtto
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establishwhetherthiswaspossible.UnlikenaturallycompetentV.cholerae,incubationon
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chitindidnotleadtodetectabletransformationinV.natriegens(datanotshown).However,
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ectopicexpressionofTfoX(eithertheendogenoustfoXgeneoronefromVibriocholerae)
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onanIPTG-inducibleplasmid(pMMB)supportedhighratesofnaturaltransformation(Fig.
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1a).ThiswastestedusingalinearPCRproductthatreplacesthegeneencodingtheDNA
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endonucleaseDnswithanantibioticresistance(AbR)marker.Thednslocuswasusedasa
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targetfortransformationassaysthroughoutthismanuscriptbecauselossofthisgenedoes
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notimpactgrowthorviabilityinrichmedium.Underoptimalconditions~1-10%ofthe
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populationhadintegratedthetransformingDNA(tDNA),whichmatchesthehighestrates
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oftransformationobservedamongcompetentspecies11(Fig.1a-c).Naturaltransformation
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ofV.natriegensrequiredverylittletransformingDNA(tDNA)(highlyefficientwitheven1
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ng/108CFU)andwasdependentonthelengthofhomologoussequencesurroundingthe
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mutation(Fig.1bandc).Thismethodcouldalsobeusedtointroducepointmutationsinto
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V.natriegens(testedwithtDNAcontaininganrpsLK43RSmRallele);however,thisactivity
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waspartiallysuppressedbythemismatchrepairsystem(Fig.1d).
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HavingdemonstratedV.natriegensisnaturallycompetent,wesoughttodetermineifwe
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couldusenaturaltransformationtoperformscarlessmultiplexgenomeeditingbynatural
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transformation(MuGENT)12.MuGENToperatesunderthepremisethatundercompetence
2
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1
inducingconditions,onlyasubpopulationofcellsistransformable.Thosecellsthatcanbe
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transformed,however,havethecapacitytotakeupandintegratemultipletDNAs12,13.Thus,
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duringMuGENT,cellsareincubatedwithtwotypesoflineartDNA;(1)aselectedproduct
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thatintroducesanantibioticresistancemarkerintothegenomeand(2)unselected
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productsthatintroducescarlessgenomeeditsofinterestatoneormoreloci.
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WefirsttestedtheabilityofMuGENTtointroduceasingleunmarkedgenomeedit(also
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knownascotransformation).Tofacilitatemeasurementofcotransformation,wenotedthis
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speciesformsopaquecoloniesonagarplates(Fig.2a),whichcouldbeduetothe
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productionofacapsularpolysaccharide.Consistentwiththis,inactivatingahomologofthe
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essentialcapsulebiosynthesisgenewbfF14resultedintheformationoftransparent
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coloniesonagarplatesandlossofexpressionofahighmolecularweightpolysaccharide
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(Fig.2aand2b).Thus,totestcotransformationweusedanunselectedproducttoreplace
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~500bpofthe5ʹendofthewbfFgenewithaprematurestopcodonandscored
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cotransformationviacolonymorphology(opaquevs.transparent)onagarplates(Fig.3a).
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WefoundthatcotransformationwasremarkablyefficientinV.natriegens(upto~80%),
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evenwithlowamounts(~25-50ng/108CFU)oftheunselectedproduct(Fig.3b).Also,
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cotransformationwith1kbflanksontheunselectedproductwaspossible,butat~6-fold
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lowerfrequenciesthanwith3kbflanks(Fig.3c).
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WenexttestedthefullmultiplexgenomeeditingcapacityofMuGENTtosimultaneously
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cotransformmultiplescarlessgenomeeditsintothegenomeinasinglestep12,15.Since
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thereisnoselectionforintegrationoftheunselectedgenomeeditsincisduringMuGENT,
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outputpopulationsarehighlyheterogeneousandindividualmutantscontainanynumber
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andcombinationofthemultiplexedgenomeedits.Also,thisprocesscanbecarriedoutin
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multipleiterativecyclestofurtherincreasethecomplexityofgenomeeditsinthe
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population(Fig.3d)12.
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Asaninitialtestofmultiplexgenomeediting,wetargeted5geneswhosemutagenesiswas
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consideredunlikelytoaffectviabilityorgrowthinLB.Thesetargetsincludedfour
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carbohydratetransporters(specificformannitol,fructose,sucrose,andtrehalose–allof
3
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
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1
whichareabsentinLB)andthednsgene.Allgenesweretargetedforinactivationby
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replacing~500bpofthe5’endofeachgenewithaprematurestopcodon.Integrationof
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genomeeditswasdeterminedbymultiplexallele-specificcolonyPCR(MASC-PCR)16(Fig.
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3e).FollowingonecycleofMuGENT,wefoundthat~70%ofthepopulationcontainedat
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least1genomeedit,with~25%ofthepopulationcontaining3-4genomeedits(Fig.3f).A
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quadruplemutantfromthisexperimentwasisolatedandwholegenomesequencingofthis
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straindidnotrevealanyoff-targetmutations.Thus,MuGENTrapidlygeneratedV.
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natriegensstrainswithmultiplelarge(0.5kb)scarlessgenomeeditsathigh-efficiency
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withoutoff-targeteffects,andcanbeusedtomakehighlycomplexmutantpopulations.
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Asaseconddemonstrationofmultiplexgenomeediting,wedemonstrateditsutilityin
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metabolicengineeringbyattemptingtorapidlyenhanceproductionofavalue-added
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chemicalinV.natriegens.Thisspeciesnaturallyaccumulateslowlevelsofthebioplastic
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precursorpoly-β-hydroxybutyrate(PHB)asastoragepolymer17.PHBisderivedfromthe
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condensationandsubsequentNADPH-dependentreductionofacetyl-CoAprecursors18.
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Thus,forourtargets,wetunedtheexpression(swapPnativeforIPTG-induciblePtac)or
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inactivatedgenesthatwehypothesizedwouldaffectNADPHand/oracetyl-CoAavailability.
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ThetargetsforpromoterswapswerethePHBsynthesisoperon(phaBAC),NADkinase
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(nadK),andtwotranshydrogenases(pntABandudhA),whiletargetsforinactivationwere
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phosphoglucoseisomerase(pgi),citratesynthase(gltA),phosphotransacetylase(pta),
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isocitratelyase(aceA),andlactatedehydrogenase(ldhA)(Fig.4a).Thus,therewere512
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possiblecombinationsforthese9genomeedits.WeperformedmultiplecyclesofMuGENT
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tointroducethesegenomeeditsintoacompetentpopulationofV.natriegens.Ateachcycle,
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theselectedproductwasdesignedtoswaptheAbRmarkeratthednslocustomaintain
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coselectionateachstep.FollowingfourcyclesofMuGENT,whichtookjust5daysto
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perform,~50%ofthepopulationhad3ormoregenomeeditsand~10%contained5+
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genomeedits(Fig.4b).ToselectmutantswithincreasedPHBproduction,wethenplated
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thisoutputpopulationontomediacontainingNilered,whichstainsPHBgranules19.Nile
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redfluorescenceontheseplateswashighlyheterogeneous,suggestingthatsome
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genotypesproducedmorePHBthantheparentisolate(Fig.4c).Anumberofhighly
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fluorescentcolonieswerepickedandthegenotypesdeterminedbyMASC-PCR.Also,PHBin
4
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1
theseselectstrainswasdirectlymeasuredbyHPLC.Cumulatively,theseanalysesrapidly
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revealedgenotypesthatproduced~100-foldmorePHBthantheparentand~3.3-fold
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morethanastrainwithjustthePtac-phaBACmutation(Fig.4d).
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Whilemanymethodsformultiplexgenomeeditinginbacterialsystemshavebeen
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described20,manyofthesearelimitedtosmallchangessuchasSNPs.MuGENT,onthe
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otherhand,canefficientlyswap,insert,orremovewholepromotersorcodingsequencesas
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demonstratedabove.Furthermore,oneofthemajorlimitationstoothermultiplexgenome
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editingmethodsisthatmutagenesismustbeperformedinstrainslackingDNArepair
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pathwaystoallowforhigh-efficiencyintegrationofgenomeedits,whichresultsinalarge
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numberofoff-targetmutations16,20.MuGENTinV.natriegensisperformedinDNArepair
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sufficientbackgrounds,thus,littletonoofftargetmutationsareintroducedduringthe
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procedureasindicatedabove.Also,unlikeothermultiplexeditingapproaches,MuGENT
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requiresnospecializedequipmentand,thus,hasthepotentialtomakemultiplexgenome
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editingcommonplace.
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Inconclusion,thisstudydemonstratesthatMuGENTisarapid,efficient,andsimpletoolfor
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engineeringtheV.natriegensgenome.Thismicrobeisalreadybeingdevelopedasan
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alternativetoE.coli,andwebelievethattheeaseandspeedofMuGENTwillextendtheuse
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ofV.natriegensasanovelchassisfordiversemolecularbiologyandbiotechnology
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applications.
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METHODS
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Bacterialstrainsandcultureconditions
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TheparentV.natriegensstrainusedthroughoutthisstudywasaspontaneousrifampicin-
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resistantderivativeofATCC140482.Foralistofallstrainsused/generatedinthisstudy,
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seeTableS1.StrainswereroutinelygrowninLB+v2salts(LBv2)3,whichisLBMillerbroth
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(BD)supplementedwith200mMNaCl,23.14mMMgCl2,and4.2mMKCl.LBv2was
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supplementedwith100μMIPTG,50μg/mLkanamycin(Kan),200μg/mLspectinomycin
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(Spec),100μg/mLrifampicin(Rif),100μg/mLstreptomycin(Sm),or100μg/mL
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carbenicillin(Carb)asappropriate.
5
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1
2
Generationofmutantstrainsandconstructs
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Mutantconstructsweregeneratedbysplicing-by-overlapextension(SOE)PCRexactlyas
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previouslydescribed21.Briefly,forthree-piecemutantconstructs(i.e.forconstructswhere
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ageneofinterestisreplacedwithanAbRcassetteorwherethenativepromoterisswapped
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foraPtacpromoter)segmentsweredesignatedUP,MIDDLE,andDOWNandcorrespondto:
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(1)UP=theupstreamregionofhomologyamplifiedwithF1andR1primers,(2)DOWN=
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thedownstreamregionofhomologyamplifiedwithF2andR2primers,and(3)MIDDLE=
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theAbRmarkerorpromoterswapfragment.Fortwo-piecemutantconstructs(i.e.for
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constructswhere~501bpofthe5’endofageneisreplacedwithastopcodon),the
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mutationofinterestisincorporatedintotheR1andF2primersusedtoamplifythe
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upstreamanddownstreamregionsofhomology,respectively.Gelpurifiedsegmentswere
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thenmixedinequalratiosandusedastemplateforaSOEPCRreactionwiththeF1andR2
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primers.AllmutantconstructsweremadeusingPhusionpolymerase.Thesewere
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introducedintotheV.natriegensgenomevianaturaltransformationasdescribedbelow.
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AllprimersusedtogeneratemutantconstructsarelistedinTableS2.
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Naturaltransformation/MuGENTassays
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StrainsharboringpMMB-tfoX(VntfoXorVctfoX)wereinducedtocompetencebygrowing
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overnight(12-18hours)inLBv2+100μg/mLcarbenicillin+100μMIPTGinarollerdrumat
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30°C.Then,~108CFUsofthisovernightculture(~3.5μL)werediluteddirectlyinto350μL
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ofinstantoceanmedium(28g/L;AquariumSystemsInc.)supplementedwith100μM
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IPTG.TransformingDNA(tDNA)wasthenaddedasindicated,andreactionswere
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incubatedstaticallyat30°Cfor5hours.Next,1mLofLBv2wasaddedandreactionswere
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outgrownat30°Cwithshaking(250rpm)for~1-2hrs.Then,reactionswereplatedfor
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quantitativecultureontomediatoselectforintegrationoftDNA(i.e.LB+drug=
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transformants)andontononselectivemedia(i.e.plainLB=totalviablecounts).
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Transformationefficiencyisshownas:transformants/totalviablecounts.
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ForMuGENT,transformationassayswereconductedexactlyasdescribedabove.Unless
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otherwisespecified,~50ngoftheselectedproductwasincubatedwithcellsalongwith
6
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
~200ngofeachunselectedproduct.Afteroutgrowth,1/10thofthereactionwasremoved
2
andplatedforMASC-PCRanalysis(describedbelow).IfmultiplecyclesofMuGENTwere
3
performed,therestofthereactionwasgrownovernightinLBv2supplementedwith100
4
μMIPTG,100μg/mLcarbenicillin(tomaintainpMMB-tfoX),andtheantibiotictoselectfor
5
integrationoftheselectedproduct.Thefollowingday,thepopulationwasthensubjectedto
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anotherroundofMuGENTasdescribedaboveusingaselectedproductcontaininga
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differentAbRmarkertomaintaincoselectionateachcycle.
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IntegrationofgenomeeditswasdetectedviaMASC-PCRexactlyaspreviouslydescribed12,
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16.Briefly,colonieswereboiledin50μLofsterilewater,vortexed,andthen2μLwereused
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astemplateina25μLPCRreaction.PCRwasconductedwithTaqpolymerase(SydLabs)
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usingamodified5XTaqbuffer:200mMTrispH8.8,100mMKCl,100mM(NH4)2SO4,30
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mMMgSO4,and1%TritonX-100.ThetotalprimerusedineachMASC-PCRreaction
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(regardlessofthenumberofmultiplexedproductsbeingdetected)was1200nM(i.e.for
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detectionof4multiplexedgenomeedits,300nMofeachgenomeedit-specificprimerpair
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wasused).Thecyclingconditionsusedwere:95°C3min;26×[95°C40s,58°C30s,72°C3
17
min];72°C3min;12°Chold.Reactionswerethenrunon2%agarosegelsandimagedwith
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GelGreendyeaccordingtomanufacturer’sinstructions(Biotium).Foralistofallprimers
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usedforMASC-PCRseeTableS2.
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21
Alcianbluestainedgels
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Topreparecelllysates,~109cellsoftheindicatedV.natriegensstrainswerepelletedand
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thenresuspendedin180μLofBufferATL(Qiagen).Then,20μLofa20mg/mLproteinase
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Kstocksolutionwasaddedtoeachreactionandincubatedat56°Cfor20mins.Samples
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werethenboiledin2XSDSPAGEsamplebufferandseparatedon4-12%SDSPAGEgels.
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Gelswerethenstainedwith0.1%AlcianBlue8GXin40%ethanol/3%aceticacidas
27
previouslydescribed22.Thegelwasthendestainedina40%ethanol/3%aceticacidand
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imagedonaBioradChemiDocMPImagingsystem.
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30
Wholegenomesequencing
7
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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
GenomicDNAwaspreppedfromstrainsandsequencinglibrarieswerepreppedvia
2
homopolymer-tailmediatedligationexactlyaspreviouslydescribed23.Single-end50bp
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readswerecollectedontheIlluminaplatform.Then,datawasanalyzedforsmallindelsand
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singlenucleotidevariantsusingCLCGenomicsWorkbenchexactlyaspreviously
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described15,24.
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7
QualitativeandquantitativeassessmentofPHBproduction
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PHBwasqualitativelyassessedinMuGENTeditedpopulationsofV.natriegensbyplating
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ontoNileredcontainingmediumwithexcessglucoseasacarbonsourceand100μMIPTG
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toinducePtac-containinggenomeedits=recipeperL:28ginstantocean,2.5gtryptone,1g
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yeastextract,20gglucose,15gagar,and1mgNilered.Fluorescenceofcolonieswas
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detectedusingaPrepOneSapphireLEDbluelightbase(475nm±30nm)andamberfilter
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(530nmlongpass)(EmbiTec).
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15
ForquantitativeassessmentofPHBlevels,theindicatedstrainsweregrownovernightin
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M9minimalmedium(BD)supplementedwith2mMMgSO4,100μMCaCl2,200mMNaCl,
17
30μMFeSO4,100μMIPTG,1%tryptone,and2%glucose.Approximately8×109cellswere
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thenpelleted,resuspendedwith50μLwaterandtransferredtopre-weighedglassscrew-
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captubes.Cellsuspensionsweredriedfor5hat80°Candthenthetubeswereweighed
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againtodeterminedrycellweights.PHBwasthenhydrolyzedandextractedascrotonic
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acidbyboilingthedriedcellsin1mlofpuresulfuricacid.Extractswerechilledoniceand
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dilutedwith4mlice-coldwater.Aliquotswerefurtherdiluted10-foldwithwater,
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centrifuged,filtered,andthencrotonicacidwasquantifiedbyHPLCasdescribed25.
24
25
ACKNOWLEDGEMENTS
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WewouldliketothankTuftsTUCFGenomicsandtheIndianaUniversityCGBforassistance
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withwholegenomesequencingofstrains.Thisworkwassupportedby
28
USNationalInstitutesofHealthGrantAI118863toABD.JBMandABDwerealsosupported
29
bytheIndianaUniversityCollegeofArtsandSciences.
30
31
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8
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
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[19]Spiekermann,P.,Rehm,B.H.,Kalscheuer,R.,Baumeister,D.,andSteinbuchel,A.(1999)
20
Asensitive,viable-colonystainingmethodusingNileredfordirectscreeningof
21
bacteriathataccumulatepolyhydroxyalkanoicacidsandotherlipidstorage
22
compounds,ArchMicrobiol171,73-80.
23
[20]Csorgo,B.,Nyerges,A.,Posfai,G.,andFeher,T.(2016)System-levelgenomeeditingin
24
25
microbes,CurrOpinMicrobiol33,113-122.
[21]Dalia,A.B.,Lazinski,D.W.,andCamilli,A.(2013)Characterizationofundermethylated
26
27
sitesinVibriocholerae,JBacteriol195,2389-2399.
[22]Mercaldi,M.P.,Dams-Kozlowska,H.,Panilaitis,B.,Joyce,A.P.,andKaplan,D.L.(2008)
28
Discoveryofthedualpolysaccharidecompositionofemulsanandtheisolationof
29
theemulsionstabilizingcomponent,Biomacromolecules9,1988-1996.
10
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
[23]Lazinski,D.W.,andCamilli,A.(2013)Homopolymertail-mediatedligationPCR:a
2
streamlinedandhighlyefficientmethodforDNAcloningandlibraryconstruction,
3
Biotechniques54,25-34.
4
[24]Seed,K.D.,Yen,M.,Shapiro,B.J.,Hilaire,I.J.,Charles,R.C.,Teng,J.E.,Ivers,L.C.,Boncy,
5
J.,Harris,J.B.,andCamilli,A.(2014)Evolutionaryconsequencesofintra-patient
6
phagepredationonmicrobialpopulations,eLife3,e03497.
7
[25]Karr,D.B.,Waters,J.K.,andEmerich,D.W.(1983)AnalysisofPoly-beta-
8
HydroxybutyrateinRhizobiumjaponicumBacteroidsbyIon-ExclusionHigh-
9
PressureLiquidChromatographyandUVDetection,ApplEnvironMicrobiol46,
10
1339-1344.
11
12
FIGURELEGENDS
13
Fig.1–NaturaltransformationofV.natriegens.(a-d)TransformationassaysofV.
14
natriegens.(a)V.natriegensstrainscontainingapMMBemptyvectororpMMBwiththe
15
tfoXgenefromeitherV.natriegens(Vn)orV.cholerae(Vc)weretransformedwith100ng
16
ofaΔdns::KanRtDNAcontaining3kbflanksofhomologyonbothsidesofthemutation(i.e.
17
3kb/3kb).TransformationassayofV.natriegenspMMB-tfoX(Vc)with(b)theindicated
18
concentrationofΔdns::KanR(3kb/3kb)tDNAor(c)5ngofΔdns::KanRtDNAcontaining
19
theindicatedamountofhomologyoneachsideofthemutation.(d)Transformationassay
20
intheindicatedstrainbackgroundswith5ngofrpsLK43RSmR(3kb/3kb)orΔdns::SpecR
21
(3kb/3kb)tDNAasindicated.AllstrainsindharborΔdns::KanRmutationsandpMMB-tfoX
22
(Vc).Alldataareshownasthemean±SDandaretheresultofatleast4independent
23
biologicalreplicates.**=p<0.01.
24
25
Fig.2–V.natriegensproducesaWbfF-dependentcapsularpolysaccharide.(a)Colony
26
morphologiesofWT(whitearrow)andΔwbfF(blackarrow)strains,whichdemonstrate
27
thephenotypesscreenedforincotransformationassays.(b)Celllysatesoftheindicated
28
strainswererunona4-12%SDSPAGEgelandstainedwiththecarbohydratestainAlcian
29
blue.ThepresenceofahighmolecularweightpolysaccharideintheWTisindicatedbya
30
redarrow.
31
11
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
Fig.3–CotransformationishighlyefficientinV.natriegens.(a)Cotransformationwas
2
testedusingaΔdns::KanR(3kb/3kb)selectedproductandanunselectedproductthat
3
deleted~500bpofthe5’endofwbfFgene.Cotransformationassayswereperformedusing
4
50ngoftheΔdns::KanR(3kb/3kb)selectedproductand(b)theindicatedamountofthe
5
ΔwbfF(3kb/3kb)unselectedproductor(c)200ngofΔwbfFunselectedproducts
6
containingtheindicatedlengthofhomologyoneachsideofthemutation.Datainbandc
7
arefromatleastfourindependentbiologicalreplicatesandshownasthemean±SD.(d)
8
SchematicofMuGENT.Theselectedproductisindicatedbyaredbox,whilemultiple
9
unselectedgenomeeditsaredepictedbydistinctgrayshapes.Sincethereisnoselectionfor
10
genomeeditsincis,outputmutantscanhaveanynumberandcombinationofthe
11
unselectedgenomeedits.(eandf)MuGENTwasperformedwith5unselectedgenome
12
edits.TheselectedproductwasΔwbfF::KanR,whiletheunselectedproductstargetedfour
13
carbohydratetransportersanddnsforinactivationbyreplacing~500bpofthe5’endof
14
eachgenewithaprematurestopcodon.(e)ArepresentativeMASC-PCRgelof24colonies
15
fromtheeditedpopulation.Thetargetsofeachgenomeeditareindicatedontheleftand
16
thepresenceofabandindicatesintegrationoftheindicatedgenomeedit.Strains
17
containing4genomeeditsareindicatedbythegreenarrows.(f)Distributionofgenome
18
editsinthepopulationdeterminedbyMASC-PCRanalysisof48randommutants.
19
20
Fig.4–MuGENTrapidlyenhancesPHBproductioninV.natriegens.(a)Theindicated
21
targetsweresubjectedtoeitherapromoterswap(top)orinactivationbyreplacing
22
~500bpofthe5’endofeachgenewithashortsequencetointroduceaprematurestop
23
codon(bottom).(b)Distributionofthe9genomeeditsinapopulationofcellsfollowing
24
fourcyclesofMuGENT.(c)Representativeimageofthemutantpoolgeneratedinbplated
25
onNileredcontainingplates,whichstainPHBgranules.Whitearrowsindicatecolonies
26
withincreasedfluorescenceintensitycomparedtotheparent.(d)PHBcontentofselect
27
MuGENToptimizedstrainsisshownasthe%ofdrycellweight(DCW).Thegenotypeof
28
eachmutantisshownbeloweachbarwhereafilledboxindicatesthepresenceofthe
29
genomeeditindicatedontheleft.Dataareshownasthemean±SDandarefromatleast2
30
independentbiologicalreplicates.*=p<0.05.
31
12
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
-
10-6
10-4
W
T
Δm
ut
S
+
10-3
W
T
Δm
ut
S
-
10-7
10-5
1k B
0. B/1
5k
B/ kB
0.
5k
B
+
10-6
10-2
10-4
B
-
pM
M
BpM tfoX
M
B- (Vc
pM tfoX )
M
B- (Vc
pM tfoX )
M
B- (Vn
)
tfo
X
(V
n)
pM
M
B
pM
M
B
100 µM IPTG: +
10-5
**
10-3
2k
10-7
10-4
10-1
3k
10-6
10-3
d
10-2
B/
10-5
10-2
c
B/
10-4
10-1
2k
10-3
100
SmR
Δdns::SpecR
Δdns::KanR(Xkb/Xkb) (pointmutant)
5ng
10-1
NS
100
3k
10-2
b
Transformation efficiency
(transformants/totalCFU)
Δdns::KanR(3kb/3kb)
Xng
0n
g
1n
g
5n
g
25
ng
10
0n
g
50
0n
g
10-1
Transformation efficiency
(transformants/totalCFU)
Transformation efficiency
(transformants/totalCFU)
a
Δdns::KanR(3kb/3kb)
100ng
Fig.1–Naturaltransforma.onofV.natriegens.(a-d)TransformaConassaysofV.natriegens.(a)V.
natriegensstrainscontainingapMMBemptyvectororpMMBwiththe4oXgenefromeitherV.
natriegens(Vn)orV.cholerae(Vc)weretransformedwith100ngofaΔdns::KanRtDNAcontaining3
kbflanksofhomologyonbothsidesofthemutaCon(i.e.3kb/3kb).TransformaConassayofV.
natriegenspMMB-4oX(Vc)with(b)theindicatedconcentraConofΔdns::KanR(3kb/3kb)tDNAor(c)
5ngofΔdns::KanRtDNAcontainingtheindicatedamountofhomologyoneachsideofthemutaCon.
(d)TransformaConassayintheindicatedstrainbackgroundswith5ngofrpsLK43RSmR(3kb/3kb)
orΔdns::SpecR(3kb/3kb)tDNAasindicated.AllstrainsindharborΔdns::KanRmutaConsandpMMB4oX(Vc).Alldataareshownasthemean±SDandaretheresultofatleast4independentbiological
replicates.**=p<0.01.
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
a
b
Fig.2–V.natriegensproducesaWbfF-dependent
capsularpolysaccharide.(a)Colonymorphologies
ofWT(whitearrow)andΔwbfF(blackarrow)
strains,whichdemonstratethephenotypes
screenedforincotransformaConassays.(b)Cell
lysatesoftheindicatedstrainswererunona4-12%
SDSPAGEgelandstainedwiththecarbohydrate
stainAlcianblue.Thepresenceofahighmolecular
weightpolysaccharideintheWTisindicatedbya
redarrow.
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
Unselectedproduct
4
3
2
1
0
60
40
20
MASC-PCR
C
1
e
60
ΔmannitolPTS
ΔtrehalosePTS
40
20
B
B
1k
B/
1k
B
0
3k
25
ng
50
n
10 g
0n
20 g
0n
50 g
0n
g
0
5
80
0
80
B/
20
Unselected
products
B/
40
Select
AbR
100
2k
60
c
3k
80
100
wbfF
Cotransformation
frequency (%)
100
Cotransformation
frequency (%)
b
dns
f
MuGENT
Selected
product
wbfF
KanR
gDNA
d
Frequency (%)
Selectedproduct
2k
a
Δdns
ΔsucrosePTS
ΔfructosePTS
Fig.3–Cotransforma.onishighlyefficientinV.natriegens.(a)CotransformaConwastestedusingaΔdns::KanR
(3kb/3kb)selectedproductandanunselectedproductthatdeleted~500bpofthe5’endofwbfFgene.
CotransformaConassayswereperformedusing50ngoftheΔdns::KanR(3kb/3kb)selectedproductand(b)the
indicatedamountoftheΔwbfF(3kb/3kb)unselectedproductor(c)200ngofΔwbfFunselectedproducts
containingtheindicatedlengthofhomologyoneachsideofthemutaCon.Datainbandcarefromatleast
fourindependentbiologicalreplicatesandshownasthemean±SD.(d)SchemaCcofMuGENT.Theselected
productisindicatedbyaredbox,whilemulCpleunselectedgenomeeditsaredepictedbydisCnctgrayshapes.
SincethereisnoselecConforgenomeeditsincis,outputmutantscanhaveanynumberandcombinaConof
theunselectedgenomeedits.(eandf)MuGENTwasperformedwith5unselectedgenomeedits.Theselected
productwasΔwbfF::KanR,whiletheunselectedproductstargetedfourcarbohydratetransportersanddnsfor
inacCvaConbyreplacing~500bpofthe5’endofeachgenewithaprematurestopcodon.(e)ArepresentaCve
MASC-PCRgelof24coloniesfromtheeditedpopulaCon.Thetargetsofeachgenomeeditareindicatedonthe
lebandthepresenceofabandindicatesintegraConoftheindicatedgenomeedit.Strainscontaining4genome
editsareindicatedbythegreenarrows.(f)DistribuConofgenomeeditsinthepopulaCondeterminedby
MASC-PCRanalysisof48randommutants.
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
a
c
Ptac
Pna.ve
phaBAC,pntAB,
nadK,andudhA
*
aceA,pta,pgi,ldhA,andgltA
Frequency (%)
100
80
60
9
8
7
6
5
4
40
20
C
1
C
2
C
3
C
4
0
3
2
1
0
# of edits
PHB content
(% of DCW)
d
b
6
5
*
*
*
4
3
2
1
0
Ptac-phaBAC
Ptac-nadK
Ptac-pntAB
Ptac-udhA
Δpta
ΔgltA
Δpgi
ΔldhA
ΔaceA
Fig.4–MuGENTrapidlyenhancesPHBproduc.oninV.natriegens.(a)The
indicatedtargetsweresubjectedtoeitherapromoterswap(top)orinacCvaConby
replacing~500bpofthe5’endofeachgenewithashortsequencetointroducea
prematurestopcodon(bofom).(b)DistribuConofthe9genomeeditsina
populaConofcellsfollowingfourcyclesofMuGENT.(c)RepresentaCveimageof
themutantpoolgeneratedinbplatedonNileredcontainingplates,whichstain
PHBgranules.Whitearrowsindicatecolonieswithincreasedfluorescenceintensity
comparedtotheparent.(d)PHBcontentofselectMuGENTopCmizedstrainsis
shownasthe%ofdrycellweight(DCW).Thegenotypeofeachmutantisshown
beloweachbarwhereafilledboxindicatesthepresenceofthegenomeedit
indicatedontheleb.Dataareshownasthemean±SDandarefromatleast2
independentbiologicalreplicates.*=p<0.05.
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
SUPPLEMENTARYTABLES
TableS1–Strainsusedinthisstudy
Strainname
Genotypeandantibiotic
resistances
WT
RifR
pMMB-tfoX(Vc)
pMMB-tfox(Vc)CarbR
pMMB-tfoX(Vn)
pMMB-tfox(Vn)CarbR
pMMB
pMMBemptyvectorCarbR
WT(Fig.1D)
pMMB-tfoX(Vc)CarbR,
Δdns::KanR
ΔmutS
pMMB-tfoX(Vc)CarbR,
Δdns::KanR,ΔmutS
ΔwbfF
pMMB-tfoX(Vc)CarbR,
ΔwbfF::KanR
MuGENTquadruple
mutant
pMMB-tfoX(Vc)CarbR,
ΔwbfF::KanR,ΔBA890_01815
(mannitoltransporter),
ΔBA890_19540(sucrose
transporter),ΔBA890_16410
(fructosetransporter),Δdns
Fig.4E,secondbar
pMMB-tfoX(Vc)CarbR,
Δdns::KanR,Ptac-phaBAC
Fig.4E,thirdbar
pMMB-tfoX(Vc)CarbR,
Δdns::KanR,Ptac-phaBAC,PtacnadK,ΔldhA
Fig.4E,fourthbar
pMMB-tfoX(Vc)CarbR,
Δdns::KanR,Ptac-phaBAC,Ptac-
Description
SpontaneousRifRderivativeV.
natriegensATCC14048thatisthe
parentisolateforallstrainsused
inthisstudy.
SAD1304containingpMMB-tfoX
(Vc),avectorcontainingthetfoX
genefromV.cholerae(VC1153)
underthecontrolofanIPTGinduciblePtacpromoter.Vectoris
derivedfrompMMB67EHandhas
aCarbRgeneforselection.
SAD1304containingpMMB-tfoX
(Vn),avectorcontainingthetfoX
genefromV.natriegens
(BA890_05980)underthecontrol
ofanIPTG-induciblePtac
promoter.Vectorisderivedfrom
pMMB67EHandhasaCarbRgene
forselection.
SAD1304containingthe
pMMB67EHemptyvector
SAD1306withΔdns::KanR
(ΔBA890_12415)
Generatedbycotransformation
intoSAD1306withΔdns::KanR
andaproducttodelete~500bpof
the5’endofthemutSgene
(BA890_12150).
IntroducedaΔwbfF::KanR
mutation(ΔBA890_01135)into
theSAD1306strainbackground.
Reference
/(strain#)
Thisstudy
(SAD1304)
Thisstudy
(SAD1306)
Thisstudy
(TND0322/
SAD1495)
Thisstudy
(TND0321/
SAD1496)
Thisstudy
(SAD1313)
Thisstudy
(TND0362/
SAD1497)
Thisstudy
(CAH509/
SAD1498)
MuGENTintoSAD1306strain
with5unselectedgenomeedits.
Thisstudy
Thisquadruplemutantwaswhole (TND0338/
genomesequencedandnooff
SAD1499)
targetmutationswereidentified.
MuGENTintoSAD1306to
enhancePHBproduction.The
straincontainsthegenomeedits
indicated.
MuGENTintoSAD1306to
enhancePHBproduction.The
straincontainsthegenomeedits
indicated.
MuGENTintoSAD1306to
enhancePHBproduction.The
Thisstudy
(TND0364/
SAD1500)
Thisstudy
(SAD1501)
Thisstudy
(SAD1502)
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
pntAB,Δpta,ΔgltA,ΔaceA
Fig.4E,fifthbar
pMMB-tfoX(Vc)CarbR,
Δdns::SpecR,Ptac-phaBAC,PtacnadK,Δpta,ΔgltA,ΔldhA
Fig.2E,sixthbar
pMMB-tfoX(Vc)CarbR,
Δdns::SpecR,Ptac-phaBAC,PtacnadK,Δpta,ΔgltA,ΔldhA,ΔaceA
straincontainsthegenomeedits
indicated.
MuGENTintoSAD1306to
enhancePHBproduction.The
straincontainsthegenomeedits
indicated.
MuGENTintoSAD1306to
enhancePHBproduction.The
straincontainsthegenomeedits
indicated.
Thisstudy
(SAD1503)
Thisstudy
(SAD1504)
TableS2–Primersusedinthisstudy
Primer
PrimerSequence(5’à 3’)*
Name
PrimersforMutantconstructs
ABD123
ABD124
BBC1264
BBC1605
BBC1607
BBC1609
BBC1265
BBC1266
BBC1610
BBC1608
BBC1606
BBC1267
DOG0246
DOG0247
DOG0248
DOG0249
BBC1255
BBC1256
BBC1257
BBC1258
BBC1552
BBC1553
BBC1554
BBC1555
BBC1347
BBC1348
BBC1349
BBC1350
DOG0353
ATTCCGGGGATCCGTCGAC
TGTAGGCTGGAGCTGCTTC
CTAACATGGCTAAGCACCTG
GCACTTCTTCGCGAATTCGC
AGTGATTGGGTCACTCATTGG
AATGAGATTCGCCTTAACCC
gtcgacggatccccggaatAGAGAACAGGTATTTCATAGTTAAAG
TC
gaagcagctccagcctacaTAATCCTCACCAATCGCGAC
TCGAGCTTTACGCCACAACG
ACACCTTGGTCGAGGTGAAG
ATAACGCAGTAGAAAGTATCCAC
ACTGGTAAGCCATAACGACC
AGGCTCGTGTTGCATGTGAG
gctaattcagtttaagcggccatCATAGTTAAAGTCTTTAAAAAGTA
TGACTT
atggccgcttaaactgaattagcATCGCTCGTACCTATCTTTATATG
TAAGGTGTCTCAAATCTCAATCTAGG
TGAGAAATTCTTTGCATCACATC
GAAGTGCTGAGTTAGGTTTTcTAGGTGTAGTAGTGTAAAC
AC
GTGTTTACACTACTACACCTAgAAAACCTAACTCAGCACTT
C
GTAGTGACGAGTTGGAGTG
GAACTGCATGAATACGTTGTTCC
gctaattcagtttaagcggcCACAGGTAAGTTCTTTTGTTTATTTC
C
GTGgccgcttaaactgaattagcCGCACCGCACCACGTGAG
GAGTATCAGCAACACAGTAACC
TAGCAACTGTTTTAGCGCTG
gtcgacggatccccggaatCTTTTATCATCATACTCATTCATTAA
AG
gaagcagctccagcctacaTGATGTATAAGCGTCATTTATTCG
GTTCCTGTCGATAAGTATTGATC
AATGTCGGCCTTCTGATTAG
Description
AmplifyMIDDLEAbR(KanR,SpecR,or
TmRcassettes)F
AmplifyMIDDLEAbR(KanR,SpecR,or
TmRcassettes)R
ΔdnsF1(3kb)
ΔdnsF1(2kb)
ΔdnsF1(1kb)
ΔdnsF1(0.5kb)
ΔdnsR1
ΔdnsF2
ΔdnsR1(0.5kb)
ΔdnsR1(1kb)
ΔdnsR1(2kb)
ΔdnsR1(3kb)
Δdns501bpF1
Δdns501bpR1
Δdns501bpF2
Δdns501bpR2
rpsLK43R(SmR)F1
rpsLK43R(SmR)R1
rpsLK43R(SmR)F2
rpsLK43R(SmR)R2
ΔmutS501bpF1
ΔmutS501bpR1
ΔmutS501bpF2
ΔmutS501bpR2
ΔwbfFF1
ΔwbfFR1
ΔwbfFF2
ΔwbfFR2
ΔwbfF501bpF1(3kb)
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
BBC1612
BBC1614
DOG0354
DOG0355
BBC1615
BBC1613
BBC1611
DOG0271
DOG0272
DOG0273
DOG0274
DOG0266
DOG0267
DOG0268
DOG0269
DOG0256
DOG0257
DOG0258
DOG0259
DOG0261
DOG0262
DOG0263
DOG0264
ABD840
ABD625
BBC1536
BBC1540
BBC1541
BBC1577
BBC1621
BBC1622
TAAACTTTATCAGCGACGTCAG
TTCAGGAACGATGTCGACAG
gctaattcagtttaagcggccatTATCATCATACTCATTCATTAAAG
TTTTAA
atggccgcttaaactgaattagcACTAATAACGTCAGTGTATACGTA
AAC
CCACGCAATGTAGTCATCAATC
GGATACGCAGCATACCTTG
TTAATTGTGCCTGAGCAAGC
AAGTAGTGATGATCCGAAGCG
gctaattcagtttaagcggccatCATAACAATTCCCCGTTCGATG
atggccgcttaaactgaattagcCTTGTATCAGCGCACCTTCTAC
ATCGTGGTAAATATCGTCAGGTAG
ATCTCGGCTTGTCTACACCAG
gctaattcagtttaagcggccatCATTGCACACCCCGATTGG
atggccgcttaaactgaattagcTATTTACCTGTTTTATTGGCGTTT
TC
TGAACTGAATCCTCGCAGG
ATGCTCGTCATCCATGGGAC
gctaattcagtttaagcggccatCATACTGATAACCTTCTGTTCCTT
AG
atggccgcttaaactgaattagcACCGCGCAAGAGATCGAAG
TTGGGTGCTTTGCTTCTCG
ATCTGAACTTAGGATACTCACATC
gctaattcagtttaagcggccatCATAACTTTGCCCACCCTGTATTG
atggccgcttaaactgaattagcTTCTTCCTGCCTGTTGGC
AGTCAGATGGCGATTGATGTG
TTAATTGCGTTGCGCTCACTGCCCGACTCCCGTTCTGGATA
ATGTTTTTTGC
CTGATGAATCCCCTAATGATTTTGG
GTAACGAACGTGTCATCAGTG
CGGGCAGTGAGCGCAACGCAATTAATGCAAGCGCACTAAT
ATGAC
CAAAATCATTAGGGGATTCATCAGAAAGAATGGAGTCGTC
AATGAATAAAG
CGACATCTTCACCAACACG
TCTGGAGAGTATGTTGGCC
cgggcagtgagcgcaacgcaattaaCCTTGTATACATATCAATTAA
TTAGTCCC
ΔwbfF501bpF1(2kb)
ΔwbfF501bpF1(1kb)
ΔwbfF501bpR1
ΔwbfF501bpF2
ΔwbfF501bpR2(1kb)
ΔwbfF501bpR2(2kb)
ΔwbfF501bpR2(3kb)
ΔBA890_01815501bp(mannitol
transporter)F1
ΔBA890_01815501bp(mannitol
transporter)R1
ΔBA890_01815501bp(mannitol
transporter)F2
ΔBA890_01815501bp(mannitol
transporter)R2
ΔBA890_19540(sucrosetransporter)
F1
ΔBA890_19540(sucrosetransporter)
R1
ΔBA890_19540(sucrosetransporter)
F2
ΔBA890_19540(sucrosetransporter)
R2
ΔBA890_16410(fructosetransporter)
F1
ΔBA890_16410(fructosetransporter)
R1
ΔBA890_16410(fructosetransporter)
F2
ΔBA890_16410(fructosetransporter)
R2
ΔBA890_03375(trehalose
transporter)F1
ΔBA890_03375(trehalose
transporter)R1
ΔBA890_03375(trehalose
transporter)F2
ΔBA890_03375(trehalose
transporter)R2
AmplifyMIDDLEPtacconstructF
AmplifyMIDDLEPtacconstructR
Ptac-phaBACF1
Ptac-phaBACR1
Ptac-phaBACF2
Ptac-phaBACR2
Ptac-pntABF1
Ptac-pntABR1
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
caaaatcattaggggattcatcagAggaggTTGCGTTTTGCAAATCGG
TGTAC
BBC1624 AGACTACGCCAAACTATACAGC
BBC1616 CTTCTTCGTCTTCAAAACGACG
cgggcagtgagcgcaacgcaattaaGCATTAAAGAGGCTTGAATCA
BBC1617
GG
caaaatcattaggggattcatcagaggaggtAATGCTATGAAAAATCC
BBC1618
ATGTAACG
BBC1619 CTGCGCTGATAATAAACAAGC
BBC1626 CACAAATAGCGAAGCTAACTG
cgggcagtgagcgcaacgcaattaaTATTTGCTTAACATTGCCTTA
BBC1627
GC
caaaatcattaggggattcatcagAggaggtTACATCATGGCGCATGT
BBC1628
AAATC
BBC1629 GTGAAAGTATTTTCGCCTTTCG
BBC1636 GACAAGTCAGAAAGTCCAGTCAC
BBC1637 gctaattcagtttaagcggccatAGACATTCGTAGAGTACCTTTGC
atggccgcttaaactgaattagcGTTATCATCAACAAGCTAAACGCA
BBC1638
C
BBC1639 GATATCAACGAGTTTGCATCTG
BBC1646 GCTAACATCAATGCGTATGCC
BBC1647 gctaattcagtttaagcggccatCAACATGGTCTTTATCCCGATG
BBC1648 atggccgcttaaactgaattagcGCACTGGCACCATACAAAAAC
BBC1649 CCTTTCTCAGACACTATCGACAC
BBC1641 AGCCTTCTTCTACATCAAGTGTG
BBC1642 gctaattcagtttaagcggccatATCCGCCATAACAATCTCCTTTG
BBC1643 atggccgcttaaactgaattagcACACTGGCGGCAATGTGTTAC
BBC1644 CAAGAGTACTACGAAGAGCTG
BBC1651 CTTGTAACACTGCCGCTAAGAG
BBC1652 gctaattcagtttaagcggccatCATGGTTCTCTCTCGAAATCATTG
BBC1653 atggccgcttaaactgaattagcATGGAAATTCTTTGCCATGATCC
BBC1654 AGTGTGTTACTTATTTGGAGGATG
BBC1631 TGAACTGCTGGCGAAAGGAC
GCTAATTCAGTTTAAGCGGCCATTGGTCTATCCCTCTTTAT
BBC1632
AATTTGC
ATGGCCGCTTAAACTGAATTAGCCTAAATGCTTACGAACTG
BBC1633
ATGAAATC
BBC1634 CGATTGAAGCTTGAAGAACAAGC
PrimersforMASC-PCR
BBC1623
ABD969
DOG0250
BBC1556
DOG0356
ATGGCCGCTTAAACTGAATTAGC
TGGTTGCCTTGTACTTTGGC
AGTGATCGAGAACAGCGG
ATAGCTACCGCGTTCAGGG
DOG0275
AGTGACGTGGATGTTCAGAC
DOG0270
DOG0260
AACCCAGTGATACCAGATGG
TATTCATCAGTGCAGCGGC
Ptac-pntABF2
Ptac-pntABR2
Ptac-nadKF1
Ptac-nadKR1
Ptac-nadKF2
Ptac-nadKR2
Ptac-udhAF1
Ptac-udhAR1
Ptac-udhAF2
Ptac-udhAR2
Δpta501bpF1
Δpta501bpR1
Δpta501bpF2
Δpta501bpR2
Δpgi501bpF1
Δpgi501bpR1
Δpgi501bpF2
Δpgi501bpR2
ΔgltA501bpF1
ΔgltA501bpR1
ΔgltA501bpF2
ΔgltA501bpR2
ΔldhA501bpF1
ΔldhA501bpR1
ΔldhA501bpF2
ΔldhA501bpR2
ΔaceA501bpF1
ΔaceA501bpR1
ΔaceA501bpF2
ΔaceA501bpR2
UniversalFprimerforallΔ501bp
genomeedits
RdetectforΔdns501bp(152bp
product)
RdetectforΔmutS501bp(402bp
product)
RdetectforΔwbfF501bp(165bp
product)
RdetectforΔBA890_01815501bp
(mannitoltransporter)(750bp
product)
RdetectforΔBA890_19540(sucrose
transporter)(650bpproduct)
RdetectforΔBA890_16410(fructose
bioRxiv preprint first posted online Mar. 31, 2017; doi: http://dx.doi.org/10.1101/122655. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
DOG0265
BBC435
BBC1551
BBC1625
BBC1620
BBC1630
BBC1640
BBC1650
BBC1645
BBC1655
BBC1656
TCTTGCATTAACTGTAAATCCACG
ACACTCTTTGGGGGCCAAAATCATTAGGGGATTCATCAG
GGTAAACCCTTTGCTGTTAAACC
CTTGAGCTCGAGAGATACG
GATAAAATTCGTGCGGCTC
AGATAATGATATGACGAGGGTC
CGAATTGGAGAAGTGTTGAAG
AACCCAGTCCCAGAATTCAAAC
GATGTTGACGCGTTTTGTTCG
GGCTTCTACGTTATTTAGTGTC
TGTTGTGAATACCCGCTAGAG
transporter)(352bpproduct)
RdetectforΔBA890_03375(trehalose
transporter)(500bpproduct)
UniversalFprimertodetectallPtac
genomeedits
RdetectforPtac-phaBAC(170bp
product)
RdetectforPtac-pntAB(400bp
product)
RdetectforPtac-nadK(260bpproduct)
RdetectforPtac-udhA(550bpproduct)
RdetectforΔpta(140bpproduct)
RdetectforΔpgi(300bpproduct)
RdetectforΔgltA(200bpproduct)
RdetectforΔldhA(450bpproduct)
RdetectforΔaceA(600bpproduct)
*LowercasenucleotidesspecifyoverlapregionsforSOEPCR