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Genome engineering: new codes, new AAs, multi-virus resistance 2-3:30pm, CLSB 521 7-Nov-2012 SB204. Thanks to: .gov || || .edu || || .org || || .com || || Read = = = = = = = = I/O = = = = = = = Write. NHGRI. NIGMS. LSRF. ArmRev.org. Oppenheimer Foundation. Azco.

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  1. Genome engineering: new codes, new AAs, multi-virus resistance 2-3:30pm, CLSB 521 7-Nov-2012 SB204 Thanks to: .gov || || .edu || || .org || || .com || || Read = = = = = = = = I/O = = = = = = = Write NHGRI NIGMS LSRF ArmRev.org Oppenheimer Foundation Azco Gen9

  2. A revolution in reading & writing DNA

  3. A terabyte per bite.

  4. 1944 1995 2008

  5. What is a minimal replicating system ? & why should we want one? 5

  6. Ribozyme-Catalyzed Transcription of an Active Ribozyme Aniela Wochner, James Attwater, Alan Coulson, Philipp Holliger, Science April 2011. Evolution & engineering of an RNA polymerase ribozyme capable of synthesizing RNAs of up to 95 nt (& synthesis of a 27 nt hammerhead endonuclease ribozyme). 6

  7. What is a minimal replicating system ? 1999 Science: Nonessential Mycoplasma genitalium protein-coding genes: 130 of 482 (+43 tRNAs) 2006 PNAS: 67 confirmed. 34 added 26: disrupted only in M. pneumoniae 37: mixed mutant pools in liquid culture 34: Limited sampling 7

  8. Essential genes of a minimal bacterium PNAS 2006 8

  9. A Whole-Cell Computational ModelPredicts Phenotype from Genotype Karr et al. Cell 2012 9

  10. A Whole-Cell Computational ModelPredicts Phenotype from Genotype Karr et al. Cell 2012 10

  11. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome Gibson, et al 2010 Science 1 bp deletion in dnaA Not detected: Sanger 1-kb, but wrong clone sent by Blue Heron. At 10-kb level, because many of the 454 reads happened to end or start near error. 11

  12. Smaller.Higher speed & accuracy requires a few extra genes(E.coli 20 min. doubling) Reconstituted ribosomes:Jewett & Church 113 kbp DNA 151 genes Pure translation: Forster & Church MSB ’05 GenomeRes.’06 Shimizu, Ueda ’01

  13. Translation 23+5S : 50S 16S : 30S E P A 13

  14. Safe Industrial Organisms Genome Engineering Rationale • Robust, rapid doubling 10-20 minutes • Non-standard amino acids • Dependence on NSAAs • DNA non-exchangeable with environment • Multi-virus resistance

  15. Testing essentiality of codons (13/64)

  16. Green= 13 codon eliminationBlue = 62 codon shuffle Lajoie, Kosuri, Mosberg, Gregg, D.Zhang 

  17. One exception of 481 codons ACT CTT GCC CTWG Vsr site highly specific mismatch repair Lajoie, Kosuri, Mosberg, Gregg, D.Zhang

  18. 4 Mbp genome CAD

  19. Divide & Conquer Genome Engineering 19

  20. Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitaliumGenome-- Gibson, et al 2008 Science 20

  21. Genome Engineering: full genome vs partial • E.coli > Yeast > Mycoplasma vs one species • Design & synthesis error consequences • 1E-7 genome transplantation efficiency • $1M vs $1 to $1k per Mb for raw DNA

  22. Applications of in vitro translation • Ribosome display • Membrane protein drug receptor studies • Personal cancer vaccines. • Labeling one protein not the whole cell. • New chemistries (e.g. mirror chirality) • Commercial Systems: Roche, Ambion, Novagen, Promega, Invitrogen, Qiagen, Stratagene, Paragon, Amersham, NEB, Sutro, EMerck Tony Forster (Vanderbilt)

  23. Mirror world : Construction of Modified Ribosomes for Incorporation of d-Amino Acids into Proteins. Hecht lab Biochemistry 2006 A highly flexible tRNA acylation method for non-natural polypeptide Synthesis. Suga lab Nature Methods 2006

  24. A vesicle bioreactor as a step toward an artificialcell assembly --Noireaux & Libchaber PNAS 2004 eGFP no vesicle a-hemolysin-eGFP in vesicle 24

  25. Genetically encoded unnatural amino acidsLiu & Schultz 2010 Ann Rev Biochem.

  26. 4. Orthogonal antibiotics(ideally inexpensive) triazole Strained cyclo-octyne Azide + hydrazone Ketone hydrazide + e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

  27. Orthogonal AA chemistry (metabolic dependence) Amide Phosphine Azide + Ketone Oxime hydroxylamine + e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

  28. 10X /year since 2005 (vs 1.5X for VLSI) Carr & Church, Nature Biotech 28

  29. Minimum: $500 per 1M oligos 4 Next-Gen Synthesis: on chips 8K Xeotron Photo-Generated Acid 12K Combimatrix Electrolytic 120K Roche, Febit Photolabile 5'protection 244K Agilent Ink-jet standard reagents Amplify pools with flanking universal primers 6 Paths to error correction 1.Hyb-Select: Tian et al. 2004 Nature 2. MutS: Carr & Jacobson 2004 NAR 3. MutHLS: Smith & Modrich 1997 PNAS 4. Endo/Exonuclease : Bang Nat Meth. 2008 5. Errase 6. Sequencing

  30. OLS bp/error SynBIOSIS Kosuri et al Nature Biotech 200-mer 250 130-mer 1300 130+Errase 5940 30

  31. 2 ways to Coalesce Next-Gen DNA reading & writing Matzas, Church, et al. (Febit,HMS) Nat. Biotech Nov 2010 "perfect" part Write Read Select pick& place sort&select PCR Oligo elute amplify PicoTitrePlate multiwell plate Roche/454 micromirror Agilent OLS Polonator Data Rolony Photorelease Array synthesis Polonatorflowcell Oligonucleotide-Design 31

  32. Integration of genome reading & writing Multiplex Automated Genome Engineering (MAGE) Wang Micromirror- Polonator 32 Terry

  33. pKO3 4 DNA homology-directed strategies E.coli #1: ds-Circle x Circle 2 step recA+ recombination + Select + counterselect Link et al J. Bact 1997 (Open-access) #2: ds-Linear x Circle 1 step 5’>3’exo Reda/E b/T + Select Zhang et al Nat.Gen 1998 Yu et al. PNAS 2000 (GeneBridges license) #3: ss-90mer x ds-Circle#4: ss-Mb x ds-Circle conjugation Costantino &Court PNAS’03 Wang et al., Nature '09 Isaacs et al., Science ‘11 CAGE MAGE

  34. 8 Optimizations for ss allele replacement Red-b >10,000x MutS 100x Co-selection 4x Oligo length 10x Lagging strand: 30x G > –13 kcal/mol 30x Phosphorothioate 3x [oligo] = 0.05-50uM Wang, et al. Nature 2009 Isaacs et al Science 2011 Ellis et al PNAS 2001

  35. Co-Selection: another factor of 4 Same replichore Cross replichore Locus that restores antibiotic resistance

  36. mismatch insertion deletion -Red ssDNA Allelic Replacement site-specific

  37. MAGEPrimase & Nuclease 5 changes per 2h 4E9 genomes/day/vial Josh Mosberg Chris Gregg Marc Lajoie 37

  38. New translation code: novel AASafety features: no functional DNA exchange multi-virus resistance 8797 22705 11802 16795 11512 22037 7016 30462 1249 2765 18894 9620 20899 12210 314 18664 30530 7490 13399 15272 9540 28866 17791 15082 4810 Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling 11569 21121 5266 7401 32080 72898 39835 34568 29581 32265 22067 12119 11924 41644 24106 46116 2771 9452 5733 38167 19820 14174 1496 26270 21050 35252 40846 24991 20813 44217 33875 54431 14901 27567 10774 36108 46524 24629 15115

  39. Why genome engineering? Multi-virus resistance Changing 13/64 codons: ACC(T), AGA(R), AGG(R), ATA(I), CCC(P), CGG(R), CTC(L), CTT(L), GCC(A), GTC(V), TAG(-), TCC(S), TGA(-) Isaacs, Lajoie, Mosberg Kosuri, Wang, Carr, et al

  40. Clinical tests of non-standard AA“orthogonal” chemistry hydrazide hydrazone Ketone + PEG-pAcPhe-hGH Ambrx, Cho, Schultz et al. higher serum stability

  41. Improving a Natural Enzyme Activity through Incorporation ofUnnatural Amino Acids -- Ugwumba et al 2010 JACS 8-11-fold improvement … in contrast to … screening hundreds of thousands of mutants with natural amino acids. 41

  42. 4 Selection technologies In vitro Clones GC-MS Sensor-selectors 42

  43. 4 ways for in vivo coupled sensors-selectors 2. tRNA-ribosome 1. riboswitches 4. mRNA binding 3. ds-DNA 43

  44. 68 Sensor-Selectors (old & new ligands) 56 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR rbsR rhaS rpoE rpoH rpoN rpoS soxS tetR torR trpR tyrR 12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6-phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3-methylxanthine Vatsan Raman

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