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Published online before print January 22, 2007
Protein Science, DOI: 10.1110/ps.062591607
 Copyright © 2007 The Protein Society
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Protein fabrication automation

J. Colin Cox, Janel Lape, Mahmood A. Sayed, and Homme W. Hellinga

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA

(RECEIVED September 28, 2006; FINAL REVISION November 25, 2006; ACCEPTED November 25, 2006)

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.

Keywords: gene assembly; automation; synthetic ORF; protein expression; fabrication

Supplemental material: see www.proteinscience.org

Reprint requests to: Homme W. Hellinga, Nanaline Building, Room #413D, Research Drive, DUMC 3711, Duke University Medical Center, Durham, NC 27710, USA; e-mail: hwh{at}biochem.duke.edu; fax: (919) 684-8885.

Abbreviations: PFA, protein fabrication automation; ORF, open reading frame; PSD, protein scaffold database; SOE, splice-overlap extension; PAGE, poly-acrylamide gel electrophoresis; SOS, synthetic ORF selection; SOX, selected ORF expression; SOS-X, synthetic ORF selection and expression.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062591607.


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