text: Protein fabrication automation. - PubMed - NCBI
  1 selected item: 17242375
  Protein Sci. 2007 Mar;16(3):379-90. Epub 2007 Jan 22.
  Cox JC1, Lape J, Sayed MA, Hellinga HW.
  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.
  PMC2203321
  10.1110/ps.062591607
  The protein fabrication automation (PFA) process.
  Protein Sci. 2007 Mar;16(3):379-390.
  Synthetic ORF assembly scheme. Lengths, locations, and orientations of oligonucleotides (half arrows) and fragments (double arrows) are shown to scale for (A) the ION-PCR assembly and (B) the SOE-PCR to assemble a full-length gene in this three-fragment scheme. Letters "s" and "a" indicate sense or antisense oligonucleotides, respectively; "A," "B," and "C" indicate subfragment identity.
  Full-length assembly for genes of varying lengths. A, ankyrin binding protein (), 519-bp ORF (two primary ION-PCR fragments are combined into the full-length gene by SOE-PCR). G, glucose binding protein from T. maritima, 927-bp ORF (three primary fragments). M, maltose binding protein from E. coli, 1125-bp ORF (four primary fragments).
  Software control for the flow of information, material, and operations. (1) User-specified amino acid sequence, sequence restrictions (e.g., forbidden restriction endonuclease sites, G:C content) and oligonucleotide and PCR fragment length limits are used by GeneFab to generate a gene scaffold assembly topology (positions and orientations of oligonucleotides). (2) User-specified reaction conditions for the ION- and SOE-PCR assemblies. (3) Allele formation is driven by mutation lists provided either by a user or by computational protein design algorithms. These are converted by GeneFab into specific oligonucleotide sequences using the scaffold topology parameters. (4) FabMgr queries the oligonucleotide database to identify duplicate oligonucleotides and generate synthesis orders for new oligonucleotide sequences. (5) New oligonucleotide microplates are added to physical and virtual inventory by FabMgr. (6) The Materials list contains all the information (reagents, oligonucleotides, source and destination locators, plasticware) required to assemble the alleles. (7) Liquid-handling robot requires specification of the robotic deck geometry (position of plates, source of water, enzyme, tips). (8) Human action is required to load the robotic deck with the correct oligonucleotides plates from the collection, plasticware (reaction plates, tips), and reagents (water, enzyme, and oligonucleotides as specified in the Materials list).
  Robotic pipetting operations. (A) Dispensing of water does not require prevention of cross-contamination or mixing. A simple multitip, multidispense fills all wells. (B) Submerged mixing for the addition of reagents to wells containing oligonucleotides requires tip discards between mixing tips. (C) Cherry picks of individual oligonucleotides requires aerial expulsion, single-tip addressability, and arbitrarily complex travel paths of the dispensing arm. Some destinations are shown for different sources (oligonucleotide libraries) and destinations (ION-PCR reaction plates). Note the use of single tips for the same oligonucleotide.
  Automated full-length gene assembly. Thirty-two alleles of Thermatoga maritima glucose binding protein (three primary ION-PCR fragments per allele) were assembled fully automatically from 96 primary ION-PCR fragments (top two panels) to yield full-length 927-bp ORFs by SOE-PCR (bottom panel).
  PFA vectors. (A) synthetic ORF selection plasmid (pSOS), used to select correctly assembled full-length ORFs from the SOE-PCR products. (B) synthetic ORF expression plasmid (pSOX), into which ORFs from pSOS are recloned for protein expression. (C) The synthetic ORF selection and expression plasmid (pSOS-X) combines the functionalities of pSOS and pSOX, obviating a recloning step. plac, lactose promoter; ptac, lactose/tryptophan fusion promoter; SD, Shine–Dalgarno sequence; His6, polyhistidine purification tag; UGA, amber stop codon; CS, cloning site; ori, origin of replication.
  Total mutation frequency per clone. Insertions, deletions, point mutations (nonsense, missense, silent) are counted as mutational events. (White bars) no genetic selection; (black bars) SOS genetic selection.
  PFA pipeline timeline: (A) Using the pSOS genetic selection plasmid, followed by recloning into the pSOX or a pET expression vector; (B) using the combined genetic selection and expression plasmid pSOS-X.