DNA Sequencing (& synthesis)

•Microbe-weather-maps: single cell sequencing @ $ 0.02/kb 30 bp microbial species then drug resistance (~1000 cells/$)•Personal IDs from dust: 30 bp from single cells then immune VDJ

Cost of DNA Synthesis & Sequencing have dropped 100 -1000X in the last few years

Polony Sequencing & Personal Genome ProjectIdentifiable traits & common (low risk) pharmacogenomics

DNA Instrumentation

Sequencing $1K/ 1E8*50bp/2days

DNA Synthesis $1K/ 1E6*160bp/day

Cell/Genome programming 4/day/well

In vivo/vitro Synthetic Biology

Initial applications

Basic Enabling Technologies

More general applications

cell-cell

signals

A-to-D & thresholds

Biosensors & 3D fabrication

Light

input

Cell-CPU interfaces

Smart materials

A synthetic multicellular system for programmed pattern formation Basu, et al. Nature 434:1130

DNA origami highly -- predictable 3D nanostructures.

Example application to NMR of membrane

proteins

Douglas, Shih, Rothemund

Programmable ligand-controlled riboregulators to monitor metabolites.

Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech.

ON

ON

OFF

In vivo Counter Designs

Xis TF4

Xis TF3

Int

Int

Xis TF5

Xis TF6

Int

Int

1

2

3

4

Riboswitch counter

Integrase bit counter

Cell-cycle counter

0

1

1

0

1

2

3

Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in vitro

Nakajima, et al. Science. 2005 Apr 15;308(5720):414-5

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin VJ, et al. Nat. Biotech 2003

Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Ro DK, et al. Nature. 2006 8

Smart therapeutics: Environmentally controlled invasion of cancer cells by engineered bacteria.

Anderson et al. J Mol Biol. 2006

Optical imaging: bacteria, viruses, and mammalian cells encoding light- emitting proteins reveal the locations of primary tumors & metastases in animals. Yu, et al. Anal. Bioanal. Chem. 2003.

accumulate in tumors at ratios in excess of 1000:1 compared with normal tissues. http://www.vionpharm.com/tapet_virulence.html

Regulated Capsule TonB, DapD

& new genetic code for safety

Synthetic Genomics • 113 kbp mini-genomes ribosome-display selection

• 4.7 Mbp new genetic codes protein drugs • 7*7 * 4.7 Mbp mini-ecosystems biosensors, surface selection, high secretors, DNA & metabolic isolation

•Top Design Utility, safety & scalability

CAD-PAM Synthesis (chip & error correction)

Combinatorics Evolution Sequence

rEcoded rE.coli v2.0

Genome synthesis pipeline

50b 15Kb 5Mb 250 Mb

Polymerase in vitro

Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church

Recombination in vivo E.coli

Error correction

Recombination in human cells

Bacterial (Artificial) Chromosomes

BACs

Human(Artificial) Chromosomes

HACs

Rationale for in vitro synthetic biology

Existing powerful molecular biology kits:DNA RNA ProteinPCR, T7 RNA pol, in vitro translation.

Build structures incompatible with cells(systems interfering with cells or vice versa)

Higher yield, accuracy,purity and/or speed.

Why new genetic codes?Precise "post-translational" modificationsNew reactive AAs (not w/ biomolecules)

e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability

AzideStrained cyclo-octyne

+

+

Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

hydrazideKetone

triazole

hydrazone

Mirror world: resistant to enzymes, parasites, predators

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

Mirror world: resistant to enzymes, parasites, predators

Alternative to ambidextrous ribosome & polymerases is chiral chemical synthesis

(Taq Polymerase is 834 AA)>174 AA African Swine Fever Virus DNA Pol X . MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKVCGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIFHFTGPVSYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELIKELGFTYRIPKKRL

Duhee Bang & Stephen Kent (2004) A One-Pot Total Synthesis of Crambin

Mini-genone: 113 kbp, 151 genes

Mini-genone: 113 kbp, 151 genes

Forster & Church (2006)Mol. Syst. Biol.

rRNAs

precursor RNAs, P RNA, mRNAs

tRNAs

smallmolecules

RNA pol

recombinase

AA-tRNAs

RNase P

chaperones

P protein

Glnmethylase

translationfactors

rProteins

ribosome

modifyingenzymes

synthetases,MFT

DNA

DNA pol

Synthesizing A Minimal Cell

DNA replication

L

L

P

T

5’ tRNA1

rRNA

5’

5’

5’

L

tRNA2 H

primer

primer

tRNA1

rRNA

primer

RNase P

self-cleavage

RNase P

31 Tagged translation proteins

Forster, Nalam, Qu, Ying & Blacklow

Synthe-tases:

Factors:

33 tRNAs with anticodon modifications

Systems Biology ofTranslation andReplication

151 Genes required.

Totally unnatural 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)

80% average yieldper unnatural coupling.

bK = biotinyllysine , mS = Omethylserine eU=2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid

3 Exponential technologies(synergistic)

Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

1E-3

1E-1

1E+1

1E+3

1E+5

1E+7

1E+9

1E+11

1E+13

1830 1850 1870 1890 1910 1930 1950 1970 1990 2010

urea

E.coli

B12

tRNA

operons

telegraph

Computation &Communication

(bits/sec)

Synthesis (daltons)

Analysis(bp/$) tRNA

.