title: Protein fabrication automation.
abstract: Facile "writing" of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: J Colin
authors.initials: JC
authors.lastname: Cox
authors.forename: Janel
authors.initials: J
authors.lastname: Lape
authors.forename: Mahmood A
authors.initials: MA
authors.lastname: Sayed
authors.forename: Homme W
authors.initials: HW
authors.lastname: Hellinga
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012371:Robotics; D012984:Software
chemical_list: D001426:Bacterial Proteins; D009841:Oligonucleotides; D011506:Proteins