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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