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8:30-9:15 am 6-Nov-2008 Forum on Science and Biothreats FAZD Lansdowne, VA
Thanks to:
Exponential technologies forreading & writing genomes
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Sequencing tracked Moore’s law (2X / 2 yr) until 2004-8 (10X / yr)
40X 98% genome $5K in 2008 ($50 for 1%?)
0.0000001
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
10
1990 1995 2000 2005 2010
$/bp
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Writing DNA '80 to '087-logs: $600 to $3 / bp doubling 14 month
2008: $500 / (244K * 60b)
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Synthetic Genomics High-Throughput Components
1. HT Chemical oligo Synthesis2. HT DNA assembly3. HT in vivo (& in vitro) systems4. HT selection 5. HT sequencing6. Integration & applications
Why not out-sourced or off-the-shelf?
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Chemical synthesis, enzymatic assembly: on/off chips
8K Xeotron Photo-Generated Acid
12K Combimatrix Electrolytic
120K Roche, Febit Photolabile 5'protection
244K Agilent Ink-jet standard reagents
Tian et al. 2004 Nature Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS
$500 per 15Mbp
Amplify pools of 50mers using flanking universal PCR primers &
3 paths to 10X error correction
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1 open-architecture hardware, software, wetware
Polonator
$150K - 2 billion beads/run
e.g.1981IBM PC
Rich Terry
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Personal Genome ProjectInherited + Environmental Genomics
VDJ-ome
TRAITS(Phenome)
Multi-tissue
Epigenome
(RNA,mC)
PERSONAL GENOME1 to 98%
One in a life-time genome + yearly ( to daily) tests
Public Health Bio-weather map : Allergens, Microbes, Viruses
Microbiome
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PGP MicrobiomeResistome: 18 Antibiotics
Dantas, Sommer, Churchunpublished
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Multiple Phyla Subsisting on 18 Antibiotics
DantasSommerChurchScience
2008
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Antibody (& TCR) VDJ regions
Roth DB et al Mol Cell Biol. 1989 9:3049 N (1-13): 14 22 13 15 10 4 5 4 2 2 3 2 1 Lefranc, The Immunoglobulin FactsBook; Janeway, Immunobiology 2001
VH*DH*NNHH*JJHH*V*JJ
46*23*N N * 6 6 * 67* 55 = > 2M combinations , 750 bp, >1E10 cells
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Maintaining clonal VDJ (H & L) mRNA phase
water-in-oil emulsion4 Encapsulation approaches
Science 309: 1728
Nature Methods 3: 551 NAR 20: 3831 Anal. Biochem. 320: 55
2 Chain co-amplification approaches
Dantas, Sommer,
Agresti, Rowat
index
NAR 20: 3831 Embleton et al. In-cell PCR from mRNA: amplifying and linking heavy and light chain V-genes within single cells.
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Time Series Vaccine ExperimentTracking human dynamic response to vaccination to 11 strains:
Hepatitis A+B, Flu A/Brisbane/59/2007 (H1N1)-like, 10/2007 (H3N2)-like, B/Florida/4/2006-like virus
Polio, Yellow fever
Meningococcus
Typhoid, Tetanus
Diptheria, Pertussis
Collect samples at
-14d, 0d,
+1d, +3d,
+7d, +14d,
+21d, +28d
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N-region lengths in circulating B-cells
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Length (aa)
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Today: 16 antigens &3 PGP-VDJ(H) combinations
Future lookup-table: (20K self + 4K
pathogen antigens) * (>2M VDJs)
ImMunoGeneTics database http://imgt.cines.fr/
Uri Laserson,
Francois Vigneault
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Genome writing example 2000-2006Dupont/Genencor: 1,3 Propanediol (7 years & $400M R&D)135 g/l at 3.5 g/l/h, 51% yield135 g/l at 3.5 g/l/h, 51% yield (90% of theoretical) from glucoseheoretical) from glucose
27 changes to 4.6 Mbp E.coliackA aldA aldB arcA crr edd gldA glpK mgsA pta ptsH ptsI yqhC Saccharomyces: DAR1 GPP2 Klebsiella: dhaB1,B2,B3,X; orfX,Y P1.5.gapA P1.6.ppc P1.6.btuR P1.6.yqhD Ptrc.galP Ptrc.glk (13 knock-outs, 8 insertions, 6 regulatory changes) http://www.patentstorm.us/patents/6432686-description.html
GlycerolDAR1 GPP2
Glycerol-3-P
- NADH- NADH
3HPA
coB12 - NADPH
yqhDdhaB1-3 1,3 propanediol
Yeast Klebsiella E.coli
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Bio-petroleum from grasses or algae Immiscible Products Facilitate Purification
• Separate from water without distillation• Decrease toxicity to producer strain• >2 million liters in 2009
aqueous
organic
0
10
20
30
40
50
60
70
80
90
100
Extracellular Intracellular
Localization
Dis
trib
uti
on
(%
to
tal)
1 2 3 4
110
3,000
50
3 monthsLeverage current infrastructure &
engines
Fatty acid derived
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Improving process yield, health, safety:
What threatens all biological systems?
What do all viruses have in common? or lack?
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PEG-pAcPhe-hGH (Ambrx) high serum stability314 TAG to TAA changes
IsaacsCharalelChurchSunWang CarrJacobsonKong Sterling
New genetic code: viral-resistance, novel amino acidsno functional GMO DNA exchange
TTT
F
30362 TCT
S
11495 TAT
Y
21999 TGT
C
7048
TTC 22516 TCC 11720 TAC 16601 TGC 8816
TTA
L
18932 TCA 9783 TAASTOP
STOP
2703 TGA STOP 1256
TTG 18602 TCG 12166 TAG 314 TGG W 20683
CTT
L
15002 CCT
P
9559 CAT
H
17613 CGT
R
28382
CTC 15077 CCC 7485 CAC 13227 CGC 29898
CTA 5314 CCA 11471 CAA
Q
20888 CGA 4859
CTG 71553 CCG 31515 CAG 39188 CGG 7399
ATT
I
41309 ACT
T
12198 AAT
N
24159 AGT
S
11970
ATC 34178 ACC 31796 AAC 29385 AGC 21862
ATA 5967 ACA 9670 AAA
K
45687 AGA
R
2896
ATG M 37915 ACG 19624 AAG 14029 AGG 1692
GTT
V
24858 GCT
A
20762 GAT
D
43719 GGT
G
33622
GTC 20753 GCC 34695 GAC 25918 GGC 40285
GTA 14822 GCA 27418 GAA
E
53641 GGA 10893
GTG 35918 GCG 45741 GAG 24254 GGG 15090
1
2
3
4
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Allele replacement strategy #3: ss-Oligonucleotide Repair
Obtain >25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, or dam)
Ellis et al. PNAS 2001Constantino & Court. PNAS 2003
DNA Replication Fork
Improved Recombination Frequency:10-4 up to 90% (> 3 log increase!)
without selection
(#1: ds-circle, #2: linear ds)
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Genome Engineering Multiplex Automation (GEMASS)in vivo homologous allele replacement (lagging SS mimics)
3 hr Cycle time. Application: 314 change for multivirus resistance
HarrisWang
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ss-oligo-genome match
2log
3 kb lacZ wt
3 kb lacZ del
~0.5 %Recomb
0
0.05
0.1
0.15
0.2
0.25
0 5 10 15 20 25 30
# contiguous bp involved
Re
co
mb
ina
tio
n E
ffic
ien
cy
MM
IN
Mismatches & InsertionsDeletions
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Design + Evolution
Lenski Citrate utilizationPalsson Glycerol utilizationEdwards Radiation resistanceIngram Lactate productionMarliere ThermotoleranceJ&J Diarylquinoline resistance
(TB)DuPont 1,3-propanediol productionTolonen Biofuel resistance (4 to 8%)Lin&Reppas Trp/Tyr (pharma precursors)
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Accelerated Evolution via GEMASS: 314 TAG to TAA
0
5
10
15
20
25
0 1 2 3 4 5 6 7
# mutations/clone
Fre
qu
en
cy
Mutation Distribution: 11 oligos, 15 cycles Mutation Distribution: 54 oligos, 45 cycles
Oligo Pool
# cycles Best Clone (98 %tile)
Maximum Fraction of mutated sites
Time
11 15 7 7/11 3 days
54 45 23 23/54 9 days
10*32 18 8 251/314 2 days
Scaling & Automation Increase Efficiency of Recombination
Wang, Isaacs, Carr, Jacobson, Church
(70X faster than 2006)
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Mirror world : resistant to enzymes, parasites, predators
Approach#1: De novo Chemical synthesis (below)
#2: Redesigned peptidyl transferase + D-AA-tRNAs (next slide)
352 AA Synthetic Dpo4 Sulfolobus DNA polymerase IV
4 peptide bonds left to construct
L-aminoacidsD-nucleotides
(current biosphere)
D-aminoacidsL-nucleotides (Mirror-biopolymers)
Duhee Bang
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113 kbp DNA 151 genes
Pure translation:Forster & Church
MSB ’05 GenomeRes.’06
Shimizu, Ueda ’01
Not minimal:High speed &
accuracy requires a few extra genes(E.coli 20 min.
doubling)
Reconstituted ribosomes:
Jewett & Church
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Bio-Engineering Safety: Minimizing Bioerror/Bioterror
• Jun-2004: A Synthetic Biohazard Non-proliferation Proposal.
• Dec 2004: DOE Synthetic Genomes: Technologies and Impact http://www.sc.doe.gov/ober/berac/SynBio.pdf
• 2005: National Science Advisory Board for Biosecurity (NSABB)
•2007: DNA synthesis and biological security Nat Biotechnol. 25:627-629.
• 2008: Sloan Foundation, MIT, JCVI Study: Options for Governance
of Synthetic Genomics
• Industry Association Synthetic Biology
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Reading DNA Writing DNA
Resistance Multi-drug resis.
via catabolism
Multi-enz resis
via new chirality
Immunity VDJ-ome Multi-virus resis.
via new codes
Instruments
Open access
SOLiD D.005
Polonator G.007
Off-chip-oligosGE-MASS
Ethics / safety open access
Personal Genome Project
Bio-security IASB
Summary