Generaterandomgeneknockoutsinnon-E.colibacteria
- Generatemutantswithimprovedgeneticsorfunction
- Identifygenesinvolvedinpathogenesis,toxicity,biofilmdevelopment
- Unravelmetabolicpathways
- Identifyessentialgenesandregulatoryelements
- Characterizenovelgenesandgenefunctions
- InsertKanselectableMarkerintogDNAandrescueclonesinE.colihostexpressingthepirgeneproduct
- 100’sofcitationsformanydifferentapplications
Applications
- Rescuecloningoftransposon-mutagenizedmicrobialgenes(Fig.1).
- Rescueofplasmidsfromnon-E.colibacteria.
AmongtheadvantagesoftransposonmutagenesisisthatthetransposonservesasamarkerthatcanbeusedtocloneandsequencetheregionofgenomicDNAthathasbeendisrupted.NothingmakesthiscloningprocesseasierthancreatingmutationsinvivowiththeEZ-Tn5™<R6Kγori/KAN-2>TnpTransposome™Kit.*Inadditiontoencodingabroadhost-rangekanamycinresistancegene,thetransposoncontainsanE.coliconditionaloriginofreplication(R6Kγori).Thepresenceofthisoriginofreplicationenablesthepropagationor"rescue"oftheregionofgenomicDNA,orplasmid,intowhichthetransposonhasbeeninserted.
First,electroporatetheTransposomeintoelectrocompetentcellsofthehighestpossIBLetransformationefficiency.ActivationoftheTransposomebyMg2+inthecellresultsintherandominsertionoftheEZ-Tn5<R6Kγori/KAN-2>Transposonintothehost'sgenomicDNA.Selecttranspositionclonesonkanamycinplatesorbyscreeningforthelossofgenefunction.
Benefits
- EasilyrecoverandpropagateplasmidsfromdiversebacterialgenerathatwillnotnormallyreplicateinE.coli.
- Simplerescuecloningprocessofmutagenizedgenesspeedsupstructure/functionstudiesandsequencing.
- RandominsertionoftransposonDNAassuresexcellentcoverageofentirebacterialchromosome.
- Rescueclonescanbesequencedbidirectionallyusingtheprimersprovidedthatarehomologoustotheendsofthetransposon.
| Figure1(clicktoenlarge).TheprocessforrescuecloningoftransposoninsertionsitesingenomicDNAusingtheEZ-Tn5™<R6Kγori/KAN-2>TnpTransposome™andTransforMax™EC100D™pir+orTransforMax™EC100D™pir-116ElectrocompetentE.coli. |
*Coveredbyissuedand/orpendingpatents,exclusivelylicensedorassignedtoEpicentre®(anIllumina®Company).
