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16-Oct-2008 DARPA Cambridge, MA

Connecting Minimal Synthetic Biology with Millibiology

Classification of replication/reproduction

How close to atomic inputs ?(e.g. autotrophs use CO2, N2, etc.)

Non-translational, non-universal construction replication:•Schulman & Winfree. ECAL 2005. Self-Replication and Evolution of DNA crystals•Sievers D, von Kiedrowski G. Nature. 1994 Self-replication of complementary nucleotide-based oligomers.

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1E-4

1E-2

1E+0

1E+2

1E+4

1E+6

1E+8

1E+10

1E+12

1E+14

1840 1860 1880 1900 1920 1940 1960 1980 2000 2020

Daltons synth

Bits/sec

Seq bp/$

3 Exponential technologies“law of accelerating returns”

Shendure et al. 2004 Nature Reviews of Genetics. Kurzweil 2002; Moore 1965 (24 month doubling til 2020)

urea B12

tRNA

telegraph

Computation &Communication

Analytic

tRNA

Synthetic chemistry

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NextGen Synthesis: off chip

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 Elledge, Hannon, et al.

$500 per 15Mbp

Application to shRNA & exon capture libraries

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Not minimal genomesHigh speed & accuracy

Mycoplasma 500 RNA & protein genesMitochondria 1500 genesMike Jewett E.coli in vitro 151 genes

• Ease of synthesis• More radical designs • Existing applications -Roche Rapid Translation System (RTS) -Ambion ActivePro In Vitro Translation Kit -Novagen, Promega, Invitrogen, Qiagen Stratagene, Paragon, Amersham

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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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CharalelChurchIsaacsSun

Wang

CarrJacobson

Kong 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

PEG-pAcPhe-hGH (Ambrx) high serum stability314 TAG to TAA changes

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