thanks to: agencourt, ambergen, atactic, beyondgenomics, caliper, genomatica, genovoxx, helicos,...
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Thanks to: Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen, ThermoFinnigan, Xeotron/Invitrogen
For more info see: arep.med.harvard.edu
DOE Wed 3-Nov-2004 11:30 AM
Analysis & Synthesis of Omes
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Systems Biology Loop
Syntheses &Perturbations
Models
Experimental designs
(Systematic)
Data
Proteasome targetingGenome engineering
Metabolic optimality
Flux & Competitive growth
DNA & RNAPolony-Seq
Synthetic Biology Tools
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DOE Synthetic Genomes: Why?Cheaper/faster "standard biology", hypothesis testing
Systems Biology: Multiple simultaneous tests
Viruses: Aid strain transfer; generate variants, new haplotypes
Anti-viral vaccines and therapeutics (including variants)
In vitro: Make products toxic in E.coli.
Microbes: Interspecific hybrids (e.g. codon usage)
Structural biology: variants
Rapid vaccine response to engineered bioterrorism.
Cell-mediated immunity + humoral.
Fix mismatch between genome analysis & synthesis
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DOE Synthetic Genomes: Why?In vitroMicrobial & Human AntimutatorsArtificial ecosystems (laboratory scales)Energy aiding pathway improvementInstrustrial production: Enzymes, SingleCellProtein, Protein-
drugs Remediation: Hybrid genomes (opt. codons), combinatorial
pathway (Maxygen & Diversa). Xylose & OilPharmaceuticals: Combinatorial synthesesNano science Combinatorial syntheses, Complex nanosystems,
more general nanoassembly (in reach of polymerases and ribosome-like factories)
Health research: 10X faster results per current $ (cost/benefit)Hypothesize & test unknown gene combinations Synthetic standards (arrays, MS, quantitation, etc)Agriculture: salt, cold, drought, pest tolerant hybrid genomes
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Motif Co-occurrence, comparative genomics, RNA clusters, and/or ChIP2-location data
P= 10-6 to 10-11
Genome Res. 14:201–208Bulyk, McGuire,Masuda,Church
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Synthetic testing of DNA motif combinations
1.3 2.4 (1.3 in argR)
1.1 1.3
0.7 2.5
0.2 1.4
1.4 3.5
RNA Ratio (motif- to wild type) for each flanking gene
Bulyk, McGuire,Masuda,Church Genome Res. 14:201–208
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Synthetic Genomes & Proteomes. Why?
• Test or engineer cis-DNA/RNA-elements •Access to any protein (complex) including post-transcriptional modifications• Affinity agents for the above.• Mass spectrometry standards, protein design• Utility of molecular biology DNA-RNA-Protein
in vitro "kits" (e.g. PCR, SP6, Roche)
Toward these goals design a chassis:• 115 kbp genome. 150 genes.• Nearly all 3D structures known.• Comprehensive functional data.
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(PURE) translation utility
Removing tRNA-synthetases, translational release-factors,RNases & proteases
Selection of scFvs specific for HBV DNA polymerase using ribosome display. Lee et al. 2004 J Immunol Methods. 284:147
Programming peptidomimetic syntheses by translating genetic codes designed de novo. Forster et al. 2003 PNAS 100:6353
High level cell-free expression & specific labeling of integral membrane proteins. Klammt et al. 2004 Eur J Biochem 271:568
Cell-free translation reconstituted with purified components. Shimizu et al. 2001 Nat Biotechnol. 19:751-5.
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in vitro genetic codes
5'
mS yU eU
UGGUUG CAG
AAC... GUU A 3'GAAACCAUG
fM TN V E
| | | | | || | |
5' Second base 3'
U
A
C
C U
mSyU
eU
A C U
G
A
0
500
1000
1500
2000
2500
3000
3500
30 40 50 60 70 80
3H-E dpm
time (min.)
fM yU mS eU E |
Forster, et al. (2003) PNAS 100:6353-7
80% average yieldper unnatural coupling.
bK = biotinyllysine , mS = Omethylserine eU=2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid
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Mirror world : enzyme, parasite, & predator resistance& access 2n diastereomers (n chiral atoms)
L-amino acids & D-ribose (rNTPs, dNTPs)
Transition: EF-Tu, peptidyl transferase, DNA-ligase
D-amino acids & L-ribose (rNTPs, dNTPs)
Dedkova, et al. (2003) Enhanced D-amino acid incorporation into protein by modified ribosomes. J Am Chem Soc 125, 6616-7
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Escherichia coli Mycoplasma 3D structureColiphage 29 DNA polymerase + +Coliphage P1 Cre recombinase - + >Coliphage Lox/Cre recombinase site - +Coliphage T7 RNA polymerase + + >Coliphage T7 RNA polymerase initiation site + + >Coliphage T7 RNA polymerase termination site + +RNase P RNA + -RNase P protein + + >RNase P site/RNA primer for DNA polymerase + +Small subunit 16S ribosomal RNA + +All 21 small subunit ribosomal proteins (1-21) + except 1,21 +Large subunit 5S ribosomal RNA + +Large subunit 23S ribosomal RNA + +Large subunit 23S rRNA G2445>m2G methylase: unknown ? -Large subunit 23S rRNA U2449>dihydroU synthetase: unknown ? -Large subunit 23S rRNA U2457>pseudoU synthetase ? -Large subunit 23S rRNA C2498>Cm methylase: unknown ? -Large subunit 23S rRNA A2503>m2A methylase: unknown ? -Large subunit 23S rRNA U2504>pseudoU synthetase ? -All 33 large subunit ribosomal proteins (1-7,9-11,13-25,27-36) + except 25, 30 +Translational initiation factor 1 + +Translational initiation factor 2 + +Translational initiation factor 3 + +Translational elongation factor Tu + +Translational elongation factor Ts + +Translational elongation factor G + +Translational release factor 1 + +Translational release factor 2 - +Translational release factor Gln methylase + +Translational release factor 3 - +Ribosome recycling factor + +33/45 Transfer RNAs (see Fig. 2) 29/33 +tRNA(I) C34>lysidine synthetase ? +tRNA(R) A34>I deaminase ? +tRNA(ASV) U34>cmo5U (=V) synthetase: unknown - -tRNA(R) U34>2sU Cys desulfurase - +tRNA(R) nm5U34 methylase ? +tRNA(R) U34>cmnm5U GTPase ? +tRNA(R) U34>cmnm5U synthetase ? +tRNA(R) cmnm5U34>nm5U,mnm5U synthetase ? -tRNA(R) G37 N1-methylase + +tRNA(RNIKM) A37>t6A N6-threonylcarbamoyl-A synthetase: unknown + -tRNA(CLFSWY) A37>i6A synthetase - +tRNA(CLFSWY) i6A37>s2i6A(ms2i6A) synthetase - +All 22 aminoacyl-tRNA synthetase subunits (20 enzymes) + except G subunit, Q + except G subunitMet-tRNA formyltransferase + +Chaperonin DnaK + +Chaperonin GroEL + +Chaperonin GroES + +
Total genes = 150Forster & Church
Oligos for 150 & 776
synthetic genes(for E.coli minigenome & M.mobile whole genome
respectively)
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Up to 760K Oligos/Chip18 Mbp for $700 raw (6-18K genes)
<1K Oxamer Electrolytic acid/base 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng , Zhou, Gulari, Gao (U.Houston) 24K Agilent Ink-jet standard reagents 48K Febit 100K Metrigen 380K Nimblegen Photolabile 5'protection Nuwaysir, Smith, Albert
Tian, Gong, Church
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Improve DNA Synthesis CostSynthesis on chips in pools is 5000X less expensive per
oligonucleotide, but amounts are low (1e6 molecules rather than usual 1e12) & bimolecular kinetics slow with square of concentration decrease!)
Solution: Amplify the oligos then release them.
10 50 10 => ss-70-mer (chip)
20-mer PCR primers with restriction sites at the 50mer junctions
Tian, Gong, Sheng , Zhou, Gulari, Gao, Church
=> ds-90-mer
=> ds-50-mer
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Improve DNA Synthesis Accuracyvia mismatch selection
Tian & Church Other mismatch methods: MutS (&H,L)
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Genome assembly
Moving forward: 1. Tandem, inverted and dispersed repeats (hierarchical assembly, size-selection and/or scaffolding)2. Reduce mutations (goal <1e-6 errors) to reduce # of intermediates 3. >30 kbp homologous (Nick Reppas)4. Phage integrase site-specific recombination, also for counters.
Stemmer et al. 1995. Gene 164:49-53;Mullis 1986 CSHSQB.
50
75
125 225 425 825 … 100*2^(n-1)
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All 30S-Ribosomal-protein DNAs(codon re-optimized)
Tian, Gong, Sheng , Zhou, Gulari, Gao, Church
1.7 kb
0.3 kb
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Improving synthesis accuracy 9-fold
MethodTotal
bp#
ClonesTrans-ition
Trans-version Deletion Addition Bp/error
Hyb selection, PCR 23641 9 7 3 5 2 1391Gel selection, PCR 24546 35 28 12 11 3 455
No selection, ligation+PCR 6093 25 6 6 22 4 160
No selection, PCR 9243 21 25 13 19 1 159
Tian & Church
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Extreme mRNA makeover for protein expression in vitro
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.
Tian & Church
20w 20m 17w 17m 16w 16m
10kd
W: wild-typeM: modified
Western blot based on His-tags
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Systems Biology Loop
Syntheses &Perturbations
Models
Experimental designs
(Systematic)
Data
Proteasome targetingGenome engineering
Metabolic optimality
Flux & Competitive growth
DNA & RNAPolony-Seq
Synthetic Biology Tools
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Why sequence?
• Cancer: mutation sets for individual clones, loss-of-heterozygosity• Pathogen "weather map", biowarfare sensors• RNA splicing & chromatin modification patterns.• Synthetic biology & lab selections• Antibodies or "aptamers" for any protein• B & T-cell receptor diversity: Temporal profiling, clinical • Preventative medicine & genotype–phenotype associations• Cell-lineage during development• Phylogenetic footprinting, biodiversity
Shendure et al. 2004 Nature Rev Gen 5, 335.
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Sequencing single molecules
Ecosystem studies really need single-cell amplification because of multiple chromosomes (& RNAs)
(Even an 80% genome coverage is better than 100 kb BACs)
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Single bacterial chromosome amplification
Ratio to unamplified hybridization along thechromosome ofEscherichia & ProchlorococusonAffymetrix chips.
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Convergence on non-electrophorectic tag sequencing methods?
Tag >400 14-26 20 100 26 bp (2-ends) EST SAGE MPSS 454 Polony-Seq • Single-molecule vs. amplified single molecule. • Array vs. bead packing vs. random• Rapid scans vs. long scans (chemically limited, 454)• Number of immobilized primers: 0: Chetverin'97 "Molecular Colonies" 1: Mitra'99 > Agencourt "Bead Polonies" 2: Kawashima'88, Adams'97 > Lynx/Solexa: "Clusters"
http://arep.med.harvard.edu/Polonator/Plone.htm
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Polony Fluorescent In Situ Sequencing Libraries
Greg PorrecaAbraham Rosenbaum
1 to 100kb Genomic1 to 100kb Genomic
M
L R
M
PCRbead
Sequencingprimers
Selectorbead
2x20bp after MmeI (BceAI, AcuI)
Dressman et al PNAS 2003 emulsion
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Cleavable dNTP-Fluorophore (& terminators)
Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65
Reduce
or
photo-cleave
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0.5% of full gel areaPolony-FISSeq: up to 2 billion beads/slide
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Polony-FISSeq: up to 2 billion beads/slideCy5 primer (570nm) ; Cy3 dNTP (666nm)
Jay Shendure
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• # of bases sequenced (total) 23,703,953
• # bases sequenced (unique) 73
• Avg fold coverage 324,711 X
• Pixels used per bead (analysis) ~3.6
• Read Length per primer 14-15 bp
• Insertions 0.5%
• Deletions 0.7%
• Substitutions (raw) 4e-5 • Throughput: 360,000 bp/min
Polony FISSeq Stats
Current capillary sequencing 1400 bp/min (600X speed/cost ratio, ~$5K/1X)
(This may omit: PCR , homopolymer, context errors)Shendure
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Systems Biology Loop
Syntheses &Perturbations
Models
Experimental designs
(Systematic)
Data
Proteasome targetingGenome engineering
Metabolic optimality
Flux & Competitive growth
DNA & RNAPolony-Seq
Synthetic Biology Tools
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.
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High accuracy special case: homopolymers (e.g. AAA, CC, etc.)
• Use "compressed" tags , ACG = ACCG=ACCCG• Quantitate incorporation • Reversible terminators• "Wobble sequencing"
All of these work.
• Maintenance of amplification fidelity using linear amplification from initial genomic fragment
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"Wobble sequencing" for homopolymers
6 positions * 16 primers * 4 dNTPs => 13 bp (paired ends)CCTCATTCTCT AA + dATP (then C, …)CCTCATTCTCT AC + dATP (then C, …). . .CCTCATTCTCTnnAA + dATP (then C, …). . . CCTCATTCTCTnnNNnnNNnnTT + dATP (then C, …)
4.5/64 bp/cycle (for wobble sequencing) vs. 2.5/4 bp/cycle (for simple sequential base-extension)