title: Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.
abstract: BACKGROUND
  Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.
  
  
  RESULTS
  Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed beta-lactamase (DeltaW290) selection cassette contains a segment inside the beta-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests.
  
  
  CONCLUSIONS
  Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway or ligase-independent cloning (LIC) .
authors.affiliation: Paul Scherrer Institut, Biomolecular Research, Villigen PSI, Switzerland.
authors.forename: Natacha
authors.initials: N
authors.lastname: Olieric
authors.forename: Melanie
authors.initials: M
authors.lastname: Kuchen
authors.forename: Sandro
authors.initials: S
authors.lastname: Wagen
authors.forename: Marion
authors.initials: M
authors.lastname: Sauter
authors.forename: Stephanie
authors.initials: S
authors.lastname: Crone
authors.forename: Sonia
authors.initials: S
authors.lastname: Edmondson
authors.forename: Daniel
authors.initials: D
authors.lastname: Frey
authors.forename: Christian
authors.initials: C
authors.lastname: Ostermeier
authors.forename: Michel O
authors.initials: MO
authors.lastname: Steinmetz
authors.forename: Rolf
authors.initials: R
authors.lastname: Jaussi
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D004247:DNA