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Biobricks: BBa_

Biobricks: BBa_. Standard Assembly. Parallel & Rolling Assembly. 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/. Free-style: FSa_.

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Biobricks: BBa_

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  1. Biobricks: BBa_ Standard Assembly Parallel & Rolling Assembly 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/

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

  3. Free-style: FSa_ Construction: Fusion PCR (Combinatorial) (non)restriction ligation Stapling Ligation independent cloning (LIC) Homologous recombination (l-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. (= 2 E.coli genomes or 20 Mycoplasmas /chip) How? 10 Mbp of oligos / $1000 chip Digital Micromirror Array ~1000X lower oligo costs 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 Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997 PNAS

  5. Organism Menu

  6. Combining two genomes in one cell: Stable cloning of the Synechocystisgenome 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 http://arep.med.harvard.edu/SBP/

  9. Full codon remapping for enhanced protein expression All small ribosomal proteins 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 Western blot based on His-tags Tian et al. Nature. 432:1050

  10. rE.coli: new in vivo genetic codes Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability 4 1 Isaacs Church Forster Carr Jacobson Jahnz Schultz 3 2

  11. 4 paths to enhanced recombination 1. Homologous (e.g. l red) 2. Integrase/recombinase 3. ZnF-nucleases 4. Meganucleases (Baker, below) • 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.

  12. Integrase-Counter Design in BioBricks Collins, Church, Endy, et al. http://parts2.mit.edu/ λ Int+ LVA p22 attP Reverse Terminator p22 attB (rev comp) λXis +AAV ECFP +AAV lHalf Bit BBa_I11060 : BBa_I11020 BBa_I11033 BBa_B0025 BBa_I11032 BBa_I11021 BBa_E0024 p22 Int+ LVA λattP Terminator λattB (rev comp) P22 Xis +AAV EYFP +AAV p22 Half Bit BBa_I11061 : BBa_I11030 BBa_I11023 BBa_B0013 BBa_I11022 BBa_I11031 BBa_E0034 • Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29, 2205-2216 • Andersen, Applied and Environmental Microbiology, 1998, 2240-2246

  13. Combinatorial Synthesis ofGenetic Networks Guet et al. Science 2002

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