10 Mbp of oligos / $300 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K.

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10 Mbp of oligos / $300 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K Nimblegen/GA Photolabile 5'protection ~1000X lower oligo costs Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction Tian et al. Nature. 432:1050 Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS

ss-Oligonucleotide Repair Obtain 25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, dam) Ellis et al. PNAS 2001 Constantino & Court. PNAS 2003 DNA Replication Fork Improved Recombination Frequency:  0.25 (> 3 log increase!)

Multiplex Automated Genome Engineering (MAGE) Wash with water & DNA pool (50) Concentrate, electroporate Resuspend, bubble, select O-ring membrane Concentrate Wang, Isaacs, Terry

Multiplex Automated Genome Engineering (MAGE) syringe pump electrically actuated valves electroporation cuvette w/ membrane filter OD sensor data acquisition system computer communication / Wang, Isaacs, Terry

rE.coli: new in vivo genetic codes TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG326TGGW20683 CTT L 15002CCT P 9559CAT H 17613CGT R CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G GTC20753GCC34695GAC25918GGC40285 GTA14822GCA27418GAA E 53641GGA10893 GTG35918GCG45741GAG24254GGG15090 Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability Isaacs Wang Church Forster Carr Jacobson