1 Biobricks: BBa_ --gca EcoRINotI t XbaI gt SpeI a NotIPstIgct-- Idempotent Vector Design for Standard Assembly of Biobricks, Tom Knight Standard Assembly Parallel & Rolling Assembly

2 Free-style: FSa_ Hydrogen production by Cyanobacteria Dutta et al. Microb Cell Fact Solar-Powered Sea Slugs Rumpho et al. Plant Physiol

3 Free-style: FSa_ Construction: Fusion PCR (Combinatorial) (non)restriction ligation Stapling Ligation independent cloning (LIC) Homologous recombination ( -red, etc.) Conjugative hierarchical assembly (no DNA preps) Post-construction: Selection & screening: Size, sequence, function Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotech 2005

4 How? 10 Mbp of oligos / $1000 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng, Zhou, Gulari, Gao (Houston) 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K Nimblegen Photolabile 5'protection Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997 PNAS ~1000X lower oligo costs (= 2 E.coli genomes or 20 Mycoplasmas /chip) Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction Digital Micromirror Array

5 Organism Menu SpeciesGeneration (hr) Genome size (Mbp) Homologous recombination Library size, comments In vitro.01 to E15 phage E-21E10 E.coli.351E-41E9, tame pathogen S. cerevisiae1.5121E-31E7, tasty Synechocystis33.51E-3?, green C.elegans48100RNAi1E6, can of worms Mouse stem cells E-6 & RNAi 1E6, cute

6 Combining two genomes in one cell: Stable cloning of the Synechocystis genome in the Bacillus genome Itaya, Fujita et al. PNAS 2005

7 Yeast recombination YAC-HAC Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation Ebersole, et al. NAR 2005

8 Genome engineering Top Design ( BioJade etc.) Detailed Design (CAD-PAM) Synthesis (chip & error correction) Sequence Combinatorics Evolution Sequence

9 Full codon remapping for enhanced protein expression RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable initially. RS-1, 3, 7, 8, 11, 14, 18, 19, 20 initially weak or undetectable. Solution: Iteratively resynthesize all mRNAs with less mRNA structure, lower %GC Tian et al. Nature. 432:1050 Western blot based on His-tags All small ribosomal proteins

10 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 Church Forster Carr Jacobson Jahnz Schultz

11 4 paths to enhanced recombination Design, activity, and structure of a highly specific artificial endonuclease. Mol Cell Oct;10(4): Chevalier BS, Kortemme T, Chadsey MS, Baker D, Monnat RJ, Stoddard BL. A conformational switch controls the DNA cleavage activity of lambda integrase. Mol Cell Jul;12(1): Aihara H, Kwon HJ, Nunes-Duby SE, Landy A, Ellenberger T. 1. Homologous (e.g. red) 2. Integrase/recombinase 3. ZnF-nucleases 4. Meganucleases (Baker, below)

12 Integrase-Counter Design in BioBricks λ Xis +AAV ECFP +AAV λ Int+ LVA BBa_E0024BBa_I11020BBa_I11021 p22 attP BBa_I11033 Reverse Terminator BBa_B0025 p22 attB (rev comp) BBa_I1103BBa_I11032BBa_I11060BBa_I11060 : P22 Xis +AAV EYFP +AAV p22 Int+ LVA BBa_E0034BBa_I11030BBa_I11031 λ attP BBa_I11023 Terminator BBa_B0013 λ attB (rev comp) BBa_I11022BBa_I11061BBa_I11061 : Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29, Andersen, Applied and Environmental Microbiology, 1998, Half Bit p22 Half Bit Collins, Church, Endy, et al.

13 Combinatorial Synthesis of Genetic Networks Guet et al. Science 2002

14.