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Biobricks: BBa_ --gca Eco. RI Not. I t Xba. I Standard Assembly gt Spe. Biobricks: BBa_ --gca Eco. RI Not. I t Xba. I Standard Assembly gt Spe. I a Not. I Pst. I gct-- 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/ 1

Free-style: FSa_ http: //www. nature. com/nature/comics/syntheticbiologycomic/ Hydrogen production by Cyanobacteria Dutta et al. Microb 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 2

Free-style: FSa_ Construction: Fusion PCR (Combinatorial) (non)restriction ligation Stapling Ligation independent cloning (LIC) Homologous 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 3

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

Organism Menu Species Generation (hr) Genome size (Mbp) Homologous recombination Library size, comments In Organism Menu Species Generation (hr) Genome size (Mbp) Homologous recombination Library size, comments In vitro . 01 to 24 . 0002 1 1 E 15 phage . 08 . 008 1 E-2 1 E 10 E. coli . 3 5 1 E-4 1 E 9, tame pathogen S. cerevisiae 1. 5 12 1 E-3 1 E 7, tasty Synechocystis 3 3. 5 1 E-3 ? , green C. elegans 48 100 RNAi 1 E 6, can of worms Mouse stem cells 24 3000 1 E-6 & RNAi 1 E 6, cute 5

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

Yeast recombination YAC-HAC Rapid generation of long synthetic tandem repeats and its application for 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 7

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

Full codon remapping for enhanced protein expression All small ribosomal proteins RS-2, 4, 5, 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 m. RNAs with less m. RNA structure, lower %GC Western blot based on His-tags Tian et al. Nature. 432: 1050 9

r. E. coli: new in vivo genetic codes Freeing 4 t. RNAs, 7 codons: r. E. coli: new in vivo genetic codes Freeing 4 t. RNAs, 7 codons: UAG, UUR, AGY, AGR e. g. PEG-p. Ac. Phe-h. GH (Ambrx, Schultz) high serum stability TTT TTC 30362 TTA 11495 TAT 22516 TCC 11720 TAC 18932 F TCT TCA 9783 S 21999 TGT Y 16601 TGC C 8816 TAA STOP 2703 TGA STOP 1256 STOP 326 TGG W 20683 17613 CGT 28382 13227 CGC 29898 4 18602 TCG 12166 TAG CTT 15002 CCT 9559 CAT CTC 15077 CCC 7485 CAC TTG L CTA 11471 CAA CCG 31515 CAG 41309 ACT 12198 AAT 34178 ACC 31796 AAC 5967 ACA 9670 AAA 37915 ACG 19624 AAG GTT 24858 GCT 20762 GAT GTC 20753 GCC 34695 GAC 14822 GCA 27418 GAA 35918 GCG 45741 GAG CTG 5314 L ATT ATC I ATA ATG GTA GTG M V CCA 71553 P H T A 1 7048 R 20888 E AGT 11970 29385 AGC AGA 14029 AGG GGT 33622 25918 GGC 40285 53641 D 7399 43719 K CGG 45687 N 39188 24159 Q CGA GGA 24254 GGG S 4859 Isaacs Church 3 21862 2896 R G 2 1692 10893 15090 Forster Carr Jacobson Jahnz Schultz 10

4 paths to enhanced recombination 1. Homologous (e. g. l red) 2. Integrase/recombinase 3. 4 paths to enhanced recombination 1. Homologous (e. g. l red) 2. Integrase/recombinase 3. Zn. F-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 11 Jul; 12(1): 187 -98. Aihara H, Kwon HJ, Nunes-Duby SE, Landy A, Ellenberger T.

Integrase-Counter Design in Bio. Bricks Collins, Church, Endy, et al. http: //parts 2. mit. Integrase-Counter Design in Bio. Bricks Collins, Church, Endy, et al. http: //parts 2. mit. edu/ l Half Bit BBa_I 11060 : p 22 Half Bit BBa_I 11061 : λ Int+ LVA p 22 att. P Reverse Terminator p 22 att. B (rev comp) BBa_I 11020 BBa_I 11033 BBa_B 0025 BBa_I 11032 p 22 Int+ LVA λ att. P Terminator λ att. B BBa_I 11030 BBa_I 11023 BBa_B 0013 (rev comp) BBa_I 11022 λ Xis +AAV ECFP +AAV BBa_I 11021 BBa_E 0024 P 22 Xis +AAV EYFP +AAV BBa_I 11031 BBa_E 0034 Lewis and Hatfull, Nuc. Acid Res. , 2001, Vol. 29, 2205 -2216 12 Andersen, Applied and Environmental Microbiology, 1998, 2240 -2246

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

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