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ElectroporationofP.aeruginosa- PreparationofElectrocompetentCells
- Inoculatea5-mlofL-brothwithcellsoftheP.aeruginosastraintobeelectrotransformed.Growcultureovernightat37°Conaroller.
- Transfer2mloftheovernightcultureto200mloffreshL-brothina1-litersidearmflask.Growthiscultureat37°Cwithshaking(200rpm)untilOD540=0.3-0.5.
- Aliquot35-40mlofculturetoeachoffoursterileOakRidgecentrifugetubes.
- Pelletthecellsbycentrifugationat7000xg(8000rpminSS-34rotor)at4°Cfor10minutes.Discardsupernatant.
- ResUSPendandwashthecellsinanequalvolume(35-40ml)ofice-coldsterile300mMsucrose.Repelletthecellsbycentrifugingasbefore.Discardsupernatant.
- Resuspendandwashthecellsonceagainin0.5volume(18-20ml)ice-cold300mMsucrose.Centrifugeasbefore.Discardsupernatant.
- Resuspendthepelletin0.01volumes(350-400µl)ice-cold300mMsucrose.Thecelldensityatthispointshouldbearound1x10(exp)11cfu/ml.
- Chillthecellsonicefor30minutes.P.aeruginosacellsthuspreparedarereadyforelectroporation.UnlikeE.coli,thesecellscannotbeelectroporatedwithhighefficencyafterbeingfrozen.
- Electroporationofthecells
.- Settheelectroporationapparatusto1.6-2.5kV,25µF.Setthepulsecontrollerto200omega.
- Add1-5µlplasmidDNAtotubescontaining40µlofelectrocompetentcellsonice.MixbyswirlingwithPipettetip.TransfertheDNAandcellstoapre-chilledelectroporationcuvette(0.2cmelectrodegap)usinganarrowpipettetip.WipeanyiceorwaterfromsidesofcuvetteusingaKimwipe.Placethecuvetteintothesamplechamber.
- Energizetheelectroporationapparatusanddeliverthepulsebypushinginbothchargingbuttonssimultaneouslyandholdinguntilashortbeepisheard.Notethetimeconstantofthepulseandtheactualvoltagedelivered.
- Removethecuvettefromthesamplechamber.Add3mlL-brothandtransferthecellstoasterilepolypropyleneculturetubeusingaglassPasturepipette.Incubateculturesfor2hoursat37°Conarollerorwithmoderateshakingtoallowforplasmidexpression.
- PlatealiquotsoftheelectroporationmixtureonL-agarplatessupplementedwiththeappropriateantibiotics.Incubateplatesat37°C.
300mMSucrose
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