automated seamless dna co-transformation cloning with direct expression vectors applying positive or negative insert selection自动化的无缝的dna克隆直接表达载体共转化应用积极或消极插入选择.pdfVIP

Olieric et al. BMC Biotechnology 2010, 10:56 /1472-6750/10/56 METHODOLOGY ARTICLE Open Access Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection 1 2 2 2 1 1 Natacha Olieric , Melanie Kuchen , Sandro Wagen , Marion Sauter , Stephanie Crone , Sonia Edmondson , Daniel Frey1 3 1 1* , Christian Ostermeier , Michel O Steinmetz , Rolf Jaussi 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 b-lactamase (ΔW290) selection cassette contains a segment inside the b-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% cor