Golden Gate Shuffling A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes.doc
GoldenGateShuffling:AOne-PotDNAShufflingMethod BasedonTypeIIsRestrictionEnzymes CarolaEngler.,RamonaGruetzner.,RomyKandzia,* icsGmbH,BiozentrumHalle,Halle,Germany Abstract ubeatleastnineseparateDNAfragmentstogetherintoan acceptorvector,with90% oftype IIsrestriction enzymes andis performedbysimplysubjectinga mix of10undigested inputplasmids (nineinsert eptorvector)toarestriction- bininginonereactionseveralfragmentsetsprepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parentaltemplates(bovinecationictrypsinogen,bovineanionictrypsinogenandhumancationictrypsinogen)eachdividedin
19,binationsinonesinglerestriction-ligationandthatexpressionscreeningofasubsetofthelibrary ,thatwecall‘Golden Gateshuffling’,isrobust,simpleandefficient,canbeperformedwithtemplatesthathavenohomology,bined withothershufflingprotocolsinordertointroduceanyvariationinanypartofagivengene. Citation: Engler C, Gruetzner R, Kandzia R, S (2009) Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enz
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