1. 1 Biobricks: BBa_ --gca EcoRINotI t XbaI gt SpeI a NotIPstIgct-- Idempotent Vector Design for Standard Assembly of Biobricks, Tom Knight 2001 http://web.mit.edu/synbio/release/docs/biobricks.pdf http://biobricks.ai.mit.edu/Assembly/ Standard Assembly Parallel & Rolling Assembly

2. 2 Free-style: FSa_ Hydrogen production by Cyanobacteria Dutta et al. Microb Cell Fact. 2005 Solar-Powered Sea Slugs Rumpho et al. Plant Physiol 2000 http://www.nature.com/nature/comics/syntheticbiologycomic/

3. 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. 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. 5 Organism Menu SpeciesGeneration (hr) Genome size (Mbp) Homologous recombination Library size, comments In vitro.01 to 24.000211E15 phage.08.0081E-21E10 E.coli.351E-41E9, tame pathogen S. cerevisiae1.5121E-31E7, tasty Synechocystis33.51E-3?, green C.elegans48100RNAi1E6, can of worms Mouse stem cells2430001E-6 & RNAi 1E6, cute

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

7. 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. 8 Genome engineering Top Design ( BioJade etc.) Detailed Design (CAD-PAM) Synthesis (chip & error correction) Sequence Combinatorics Evolution Sequence http://arep.med.harvard.edu/SBP/

9. 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. 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 28382 CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S 11970 ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G 33622 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 1 2 3 4

11. 11 4 paths to enhanced recombination Design, activity, and structure of a highly specific artificial endonuclease. Mol Cell. 2002 Oct;10(4):895-905. 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. 2003 Jul;12(1):187-98.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. 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, 2205-2216 Andersen, Applied and Environmental Microbiology, 1998, 2240-2246 Half Bit p22 Half Bit Collins, Church, Endy, et al. http://parts2.mit.edu/

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

14. 14.