Applications

• CloningofunstableDNAsequencesorthoseexpressingtoxicproteins.

CopyCutter™EPI400™E.coli*cellsweredevelopedtosignificantlylowerthecopynumberofawidevarietyofcommonvectorssothatyoucanmorereADIlycloneunstableDNAsequences.DNAthatisunstableathigh-copynumberoftencodesforaproteinthatinhibitscellgrowthorcontainsAT-andGC-richsequencesorsequenceswithstrongsecondarystructure(Fig.2).¹

TheCopyCutterEPI400celllinewasderivedfromEpicentre'shigh-transformationefficiencyphageT1-resistantTransforMAX™EC100™-T1^RE.colistrainbymanipulatingagenethatcontrolsthecopynumberofvectorscontainingColE1orpMB1originsofreplication(e.g.,pUC-andpET-typevectors).Thisconstitutivelyexpressedgene,pcnB(plasmidcopynumber),wasdeletedfromtheTransforMAXEC100strainandreplacedwithamodifiedpcnBgenethatislinkedtoaninducIBLepromoter,creatingtheCopyCutterEPI400strain.

ThecopynumberofColE1-typevectorsintheCopyCutterEPI400straincomparedtotheparentalTransforMAXEC100strainisapproximately4-to25-foldlower,dependingonthevector.Moreover,ashortincubationinthepresenceoftheCopyCutterInductionSolutioncanincreasethecopynumberofthevectortoimproveplasmidyields(Fig.3).

Figure1.Copy-numberofColE1-typeplasmidsisloweredupto25-foldinCopyCutter™EPI400™E.colicells.LanesC,TransforMax™EC100™cells;LanesUandI,uninducedorinducedCopyCutterEPI400cells.DNAextractsfromthesamenumberoflysedcells(basedonOD₆₀₀)wereloadedperlane.

Figure2.DNAinsertsencodingtoxicgeneproductsweresuccessfullyclonedintohigh-copy-numbervectorsusingCopyCutter™EPI400™E.colicells.Aftersequencing,thefull-lengthacpPclonesinTransforMAX™EC100™cellswerefoundtocontainmultiplepointmutations.	Figure3.UninducedCopyCutter™EPI400™E.colicellscontainingaregBclone(laneU)areinducedtohigher-copynumber(laneI)usingtheCopyCutterInductionSolution.CrudeextractsofplasmidDNAwerepreparedfromcellsgrowninselectivemediaandanalyzedbyagarosegelelectrophoresis.Approximatelythesamenumberoflysedcells(basedonA600)wereloadedperlane.

Benefits

• Maintainclonesatlow-copynumber,theninducetohighercopynumberforimprovedplasmidyield.
• Hightransformationefficiencywithclonesofallsizes.
• tonAforresistancetobacteriophagesT1andT5.
• lacZΔM15forblue/whitescreeningofrecombinants.
• Restrictionminus[mcrA,Δ(mrr-hsdRMS-mcrBC)]genotypeenablesefficientcloningofmethylatedDNA.
• Endonucleaseminus(endA1)toensurehighyieldsofDNA.
• Recombinationminus(recA1)forgreaterstabilityoflargeclonedinserts.

Genotype

F^-mcrAΔ(mrr-hsdRMS-mcrBC)Φ80dlacZΔM15ΔlacX74recA1endA1araD139Δ(ara,leu)7697galUgalKλ^-rpsL(Str^R)nupGtrfAtonApcnB4dhfr

CopyCutterEPI400ElectrocompetentE.coli

• Transformationefficiencyof>1x10¹⁰cfu/µgofpUC19.

CopyCutterEPI400ChemicallyCompetentE.coli

• Transformationefficiencyof>1x10⁷cfu/µgofpUC19.

Reference

1. Haskins,D.(2004)EpicentreForum11(5),6.

*Coveredbyissuedand/orpendingpatents.