Recombineering/Lambda redmediated gene replacement

Single-geneknockoutsusingλredsystem,adaptedfromDatsenkoandWannerpaper.ThegoalofthisprotocolwastocreateanendA(endonucleaseI)knockout,butobviouslyitcanbeadaptedtoanygene.Theknocked-outgeneisreplacedwithanantibioticresistancegene,usuallyforkanamycinorchloramphenicol.Inthisexample,thetargetstrainwasalreadykanamycinresistant,sothechloramphenicolresistancegenewasused.

Notacompletelist—seeprotocolfordetails,orupdatethislist--smd20:20,5March2007(EST)

• plasmids
  • pKD46
  • pKD3(chloramphenicol)
  • pKD4(kanamycin)
  • pCP20(optional)
• reagents
  • L-arABInose
• equipment
  • incubators(30°Cand37°C)
  • electroporator

• Generaloutline
  • GrowuppKD46,pKD3,andpCP20inhoststrains
  • Performminiprepstoextractplasmids
  • TransformpKD46intotargetstrain,plateoutonLB-ampplates
  • PCRamplifylinearfragmentfrompKD3orpKD4usingoligosAandB(seebelowfordesign)
  • Maketargetstrain(nowmaintainingpKD46)electrocompetentbygrowingat30°CwithL-arabinose
  • ElectropratelinearDNAintoelectrocompetentcells
  • Growat37°Conchloramphenicolplates
  • PCRverifythedeletionwitholigosCandD(seebelowfordesign)
• Detailedprocedure
• Day0:Startovernightculture
  • Startovernightcultureofstraincontaininggenetoknockout.
• Day1:Preparationandtransformationofcompetentcells
  • Makenewglycerolstockofovernightstrain(grownfromsinglecolony)
  • Add300μLovernightcultureto30mLLBmedium(1:100dilution)
  • Checkculturedensityevery30minutesstartingat+1hour;growtoOD₆₀₀of0.3to0.4
  • OD₆₀₀measurementsofK91
    • +2:00hrs:0.06
    • +2:45hrs:0.3008
  • Spinat2500rcffor10minutesat4°Cintwo50mLcentrifugetubes(JA-20rotor)
  • Decantsupernatant,discard
  • ResUSPendeachpelletin5mLicecoldtransformationbuffer;swirlorPipettegentlytomix
    • TransformationBuffer
    • 10mMPipes
    • 15mMCaCl₂
    • 250mMKCl
    • TitratetopH6.7beforeaddingMnCl₂
    • 55mMMnCl₂
    • Filtersterilize
  • Incubateonicefor10min
  • Spinat2500rcffor10minutesat4°C
  • Decantsupernatant,discard
  • Resuspendeachpelletin1.25mLicecoldtransformationbuffer
  • Combineresuspendedpelletsinsingletube
  • Remove400μLforimmediatetransformation
  • AddDMSOtoafinalconcentrationof7%(160μL).DriptheDMSOslowlyintothecellsuspension,withconstantswirlingbyhand.
  • Incubateonicefor10min
  • Aliquot400μLeachintofive1.5mLtubes
  • Storein-80°Cfreezer.
  • TransformstrainwithpKD46andgrowonLB-ampplateat30°C
    • Preparefourtubeswith0,1ng,10ng,and100ngpKD46plasmidDNA
    • Add100μLofcompetentcellmixtoeachtube
    • Incubateonice30min
    • Heatshock30secondsat42°C
    • Incubateonice2min
    • Spreadall100μLonLB-amp-kanplate
    • Incubateat30°Covernight
  • U45endA—pKD3[Cat]—D45endAfragmentischloramphenicolcassettewithFRTsequences,flankedby45bpupstreamanddownstreamofendA.
  • PCRU45endA—pKD3[Cat]—D45endAfrompKD3andverifyongel
    • PCRprogram:95°C7min→35*[94°C15s→50°C30s→72°C90s]

Contents	Concentration	Volume
pKD3template	45ng/μL	0.5μL
forwardprimer	10μM	5μL
reverseprimer	10μM	5μL
10xKODbuffer	-	10μL
dNTP	2mM	10μL
MgSO4	25mM	4μL
KODpolymerase		2μL
dH2O	-	64.5μL

• • Maketen10μg/mlchloramphenicolplatesandten25μg/mlchloramphenicolplates

• Day2
  • Make1MstockofL-arabinose
    • MWofL-arabinoseis150.13
    • Add1501.3mgofL-arabinoseto8.5gdH₂Otomake1Mstock
  • Retrieveplatesfromincubator
  • Checkresultsfromthetransformation
    • 1ngpKD46transformationyieldedabout10colonies
    • 10ngpKD46transformationyieldedabout100colonies
    • 100ngpKD46transformationyieldedseveralhundredcolonies
  • Picksomecoloniesandgrowat30°Cin2mLLB+50μg/mLAmp
    • add50μL10mg/mLAmpicillinstock
  • Includeenoughsamplesfortwoconditions:+/-L-arabinoseinduction
  • WhenOD₆₀₀ofcells(+pKD46)reaches0.1,addL-arabinosetoconcentrationof10mMtoinducepKD46λ-redexpression
    • add20μL1ML-arabinoseto2mLculture
  • Continuetogrowat30°CtoOD₆₀₀=0.4
  • Aliquot1mLeachintotwo1.5mLcentrifugetubes
  • Chillcellsinice-waterbath10minutes
  • Centrifuge10minat4000rcf4°C
  • Pipetteoffsupernatantandresuspendpelletsin1mLice-colddH₂O
  • Centrifuge10minat4000rcf4°C
  • Resuspendpelletin50μLdH₂O
  • Forelectroporationstep,include2conditions:+/-PCRfragment
  • Chillelectroporationcuvettesfor5minutesonice
  • Add5pgto0.5μgPCRamplifiedDNAtocells
  • Setelectroporationapparatusto2.5kV,25μF.Setthepulsecontrollerto200ohms
  • Placethecuvetteintothesamplechamber
  • Applythepulsebypushingthebutton
  • Removethecuvette.Immediatelyadd1mLLBmediumandtransfertoasterileculturetube
  • Incubate60-120minwithmoderateshakingat37°C
  • PlatealiquotsofthetransformationcultureonLBplatessupplementedwithchloramphenicol(10μg/mL,25μg/mL)

Designingnecessaryprimers

• FirstlookatthesequenceoftheplasmidcontainingtheresistanceMarkeryouwishtoswapinforyourtargetgene.
  • NCBIsequenceviewer:pKD3
    • primingsite1:GTGTAGGCTGGAGCTGCTTC
    • primingsite2:GGACCATGGCTAATTCCCAT
    • primingsite2reversecomplement:ATGGGAATTAGCCATGGTCC

• pKD3Catsequence(1034bases)

GTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGACCATGGCTAATTCCCAT

• Next,findthesequence(andcontextsequence)ofthegeneyouwishtoremove
  • Inthisexample,IfoundthesequenceandcontextforendA(808basestotal)
    • MG1655_m56_ABE-0009661+50bpupstream+50bpdownstream

CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTGATGTACCGTTATTTGTCTATTGCTGCGGTGGTACTGAGCGCAGCATTTTCCGGCCCGGCGTTGGCCGAAGGTATCAATAGTTTTTCTCAGGCGAAAGCCGCGGCGGTAAAAGTCCACGCTGACGCGCCCGGTACGTTTTATTGCGGATGTAAAATTAACTGGCAGGGCAAAAAAGGCGTTGTTGATCTGCAATCGTGCGGCTATCAGGTGCGCAAAAATGAAAACCGCGCCAGCCGCGTAGAGTGGGAACATGTCGTTCCCGCCTGGCAGTTCGGTCACCAGCGCCAGTGCTGGCAGGACGGTGGACGTAAAAACTGCGCTAAAGATCCGGTCTATCGCAAGATGGAAAGCGATATGCATAACCTGCAGCCGTCAGTCGGTGAGGTGAATGGCGATCGCGGCAACTTTATGTACAGCCAGTGGAATGGCGGTGAAGGCCAGTACGGTCAATGCGCCATGAAGGTCGATTTCAAAGAAAAAGCTGCCGAACCACCAGCGCGTGCACGCGGTGCCATTGCGCGCACCTACTTCTATATGCGCGACCAATACAACCTGACACTCTCTCGCCAGCAAACGCAGCTGTTCAACGCATGGAACAAGATGTATCCGGTTACCGACTGGGAGTGCGAGCGCGATGAACGCATCGCGAAGGTGCAGGGCAATCATAACCCGTATGTGCAACGCGCTTGCCAGGCGCGAAAGAGCTAACCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC

• Constructprimersthathaveinternaloverlapwiththeresistancemarker(pKD3)andexternaloverlapwiththetargetknockoutgene(endA).
  • Forwardprimer:A
    • CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTGGTGTAGGCTGGAGCTGCTTC
  • Reverseprimer:B
    • GGTTGTACGCGTGGGGTAGGGGTTAACAAAAAGAATCCCGCTAGTGTAGGATGGGAATTAGCCATGGTCC

• Constructprimersthatonlyflankthetargetgene(endA)forPCRverification
  • Forwardprimer:C
    • CCAAAACAGCTTTCGCTACGTTGCT(25bases)
  • Reverseprimer:D
    • GGTTGTACGCGTGGGGTAGGGGTTA(25bases)

• Figureoutthesequenceandsizeofwhatyoushouldexpectifeverythingworks.Inthiscase,it""sCatinsertedintoendAflankingregion(1132basestotal)

CCAAAACAGCTTTCGCTACGTTGCTGGCTCGTTTTAACACGGAGTAAGTGGTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCATTTAAATGGCGCGCCTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTATTCATTAAGCATCTGCCGCATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGTAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGACAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTAGGCGCGCCGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTAAGGAGGATATTCATATGGACCATGGCTAATTCCCATCCTACACTAGCGGGATTCTTTTTGTTAACCCCTACCCCACGCGTACAACC

Literature

1. DatsenkoKAandWannerBL.One-stepinactivationofchromosomalgenesinEscherichiacoliK-12usingPCRproducts.ProcNatlAcadSciUSA2000Jun6;97(12)6640-5.doi:10.1073/pnas.120163297pmid:10829079.