Transcript ICSB3: DRPM Measures

Exponential technologies for
reading & writing genomes
8:30-9:15 am 6-Nov-2008 Forum on Science and Biothreats FAZD Lansdowne, VA
Thanks to:
1
Sequencing tracked Moore’s law
(2X / 2 yr) until 2004-8 (10X / yr)
10
1
0.1
0.01
$/bp
0.001
0.0001
0.00001
0.000001
0.0000001
1990
1995
2000
2005
2010
40X 98% genome $5K in 2008 ($50 for 1%?)
2
Writing DNA '80 to '08
7-logs: $600 to $3E-5 / bp
doubling 14 month
2008: $500 / (244K * 60b)
3
Synthetic Genomics High-Throughput Components
1.
2.
3.
4.
5.
6.
HT Chemical oligo Synthesis
HT DNA assembly
HT in vivo (& in vitro) systems
HT selection
HT sequencing
Integration & applications
Why not out-sourced or off-the-shelf?
4
Chemical synthesis, enzymatic assembly: on/off chips
$500 per 15Mbp
8K Xeotron Photo-Generated Acid
12K Combimatrix Electrolytic
120K Roche, Febit Photolabile 5'protection
244K Agilent Ink-jet standard reagents
Amplify pools of 50mers using
flanking universal PCR primers &
3 paths to 10X error correction
Tian et al. 2004 Nature
Carr & Jacobson 2004 NAR
Smith & Modrich 1997 PNAS
5
1 open-architecture hardware, software, wetware
e.g.
1981
IBM
PC
$150K - 2 billion beads/run
Polonator
Rich Terry
6
Personal Genome Project
Inherited + Environmental Genomics
One in a life-time genome + yearly ( to daily) tests
Public Health Bio-weather map : Allergens, Microbes, Viruses
PERSONAL
GENOME
1 to 98%
Multitissue
Epigenom
e
(RNA,mC)
VDJ-ome
TRAITS
(Phenome)
Microbiome
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PGP Microbiome-Resistome: 18 Antibiotics
Dantas, Sommer, Church
unpublished
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Multiple Phyla Subsisting on 18 Antibiotics
Dantas
Sommer
Church
Science
2008
9
Antibody (& TCR) VDJ regions
VH*DH*NH*JH*Vkl*Jkl
46*23*N * 6 * 67* 5 = > 2M combinations , 750 bp, >1E10 cells
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
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Maintaining clonal VDJ (H & L) mRNA phase
water-in-oil emulsion
4 Encapsulation approaches
Dantas, Sommer,
Science 309: 1728
Nature Methods 3: 551
NAR 20: 3831
Anal. Biochem. 320: 55
Agresti, Rowat
2 Chain co-amplification approaches
NAR 20: 3831 Embleton et al. In-cell
PCR from mRNA: amplifying and
linking heavy and light chain V-genes
within single cells.
11
index
Time Series Vaccine Experiment
Tracking 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)
Uri Laserson,
Francois Vigneault
ImMunoGeneTics database
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http://imgt.cines.fr/
Genome writing example 2000-2006
Dupont/Genencor: 1,3 Propanediol (7 years & $400M R&D)
135 g/l at 3.5 g/l/h, 51% yield (90% of theoretical) from glucose
27 changes to 4.6 Mbp E.coli
ackA 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
Yeast
DAR1
Glycerol-3-P
- NADH
Klebsiella
GPP2
yqhD
dhaB1-3
Glycerol
E.coli
3HPA
coB12
- NADPH
1,3
propanediol
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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
100
organic
Distribution (% total)
90
80
70
3,000
60
50
40
30
20
10
0
aqueous
Extracellular
Intracellular
50
Localization
Fatty acid derived
10
1
1
Leverage current
infrastructure &
engines
2
3
4
3 months
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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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New genetic code: viral-resistance, novel amino acids
no functional GMO DNA exchange
PEG-pAcPhe-hGH (Ambrx) high serum stability
314 TAG to TAA changes
TTT
TTC
F
TTA
30362
TCT
11495
TAT
22516
TCC
11720
TAC
18932
TCA
9783
S
21999
TGT
Y
16601
TGC
C
8816
TAA
STOP
2703
TGA
STOP
1256
STOP
314
TGG
W
20683
17613
CGT
28382
13227
CGC
29898
20888
CGA
39188
CGG
7399
24159
AGT
11970
29385
AGC
45687
AGA
14029
AGG
43719
GGT
25918
GGC
4 18602
TCG
12166
TAG
CTT
15002
CCT
9559
CAT
CTC
15077
CCC
7485
CAC
CTA
5314
CCA
11471
CAA
71553
CCG
31515
CAG
41309
ACT
12198
AAT
34178
ACC
31796
AAC
5967
ACA
9670
AAA
37915
ACG
19624
AAG
24858
GCT
20762
GAT
20753
GCC
34695
GAC
TTG
CTG
L
L
ATT
ATC
I
ATA
ATG
M
GTT
GTC
V
P
T
H
Q
N
K
D
1
A
7048
R
S
R
4859
3 21862
2
2896
1692
33622
Isaacs
Charalel
Church
Sun
Wang
Carr
Jacobson
Kong
Sterling
40285
G
GTA
14822
GCA
27418
GAA
GTG
35918
GCG
45741
GAG
E
53641
GGA
10893
24254
GGG
15090
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Allele replacement strategy #3: ss-Oligonucleotide Repair
(#1: ds-circle, #2: linear ds)
DNA Replication Fork
Ellis et al. PNAS 2001
Constantino & Court. PNAS 2003
Obtain >25% recombination efficiency in
E. coli strains lacking mismatch repair
genes (mutH, mutL, mutS, uvrD, or dam)
Improved Recombination Frequency:
10-4  up to 90% (> 3 log increase!)
without selection
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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
Harris
Wang
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ss-oligo-genome match
Recombination Efficiency
0.25
Deletions
Mismatches & Insertions
3 kb lacZ wt
0.2
0.15
MM
IN
0.1
0.05
3 kb lacZ del
0
0
5
10
15
20
25
30
2log
~0.5 %
Recomb
# contiguous bp involved
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Design + Evolution
Lenski
Palsson
Edwards
Ingram
Marliere
J&J
DuPont
Tolonen
Lin&Reppas
Citrate utilization
Glycerol utilization
Radiation resistance
Lactate production
Thermotolerance
Diarylquinoline resistance (TB)
1,3-propanediol production
Biofuel resistance (4 to 8%)
Trp/Tyr (pharma precursors)
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Accelerated Evolution via GEMASS: 314 TAG to TAA
Mutation Distribution: 11 oligos, 15 cycles
Mutation Distribution: 54 oligos, 45 cycles
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Frequency
20
15
10
5
0
0
1
2
3
4
5
6
7
# mutations/clone
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
(70X faster than 2006)
Wang, Isaacs, Carr, Jacobson, Church
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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-aminoacids
D-nucleotides
(current biosphere)
D-aminoacids
L-nucleotides
(Mirror-biopolymers)
Duhee
Bang
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113 kbp DNA 151 genes
Not minimal:
High speed &
accuracy requires
a few extra genes
(E.coli 20 min.
doubling)
Reconstituted
ribosomes:
Jewett & Church
Pure translation:
Forster & Church
MSB ’05
GenomeRes.’06
Shimizu, Ueda ’01
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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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Summary
Reading DNA
Writing DNA
Immunity
Multi-drug resis.
via catabolism
VDJ-ome
Instruments
Open access
Ethics / safety
open access
SOLiD D.005
Polonator G.007
Personal Genome
Project
Multi-enz resis
via new chirality
Multi-virus resis.
via new codes
Off-chip-oligos
GE-MASS
Resistance
Bio-security
IASB
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