17-Apr-2008 3:30-3:45 PMAvogadro-Scale Computing MIT Bartos E15

Thanks to:

Greedy Algorithms in the Libraries of Biology

PGP

Present26720 km/h4500m

pm-Mm3oK2000 yr

Is biology optimal?

Human PastLocomotion 50 km/hOcean depth 75mVisible .4-.7 Cold 0oCMemory 20 yr

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 technologies1 to 18 month doubling times

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

urea B12tRNA

telegraph

Computation &Communication

Analytic tRNA

Synthetic chemistry

human

Gb chips

Avogadro scale, >>Yottaflops (from CMOS to sea moss)

Ultra-parallel 1038 units (lab libraries:108 to 1015 25mers)

AdaptableEvolution (years), Immune (days), Neural (seconds)

Thermodynamic limit 2x1019 op/J (irreversible) 3 x1020 for polymerase (1010 for current computers)

Memory density: Neural: (1012 op/s & 106 bits)/mm3, DNA: (103 op/s & 1 bit)/nm3

Error rate: DNA: 10-9 ; RNA/protein: 10-4

Biofuel: 4x107 J/kg (~=$) Adleman 1994

DNA error rates

Ellis et al. PNAS 2001Constantino & Court. PNAS 2003

DNA Replication Fork

3. Mismatch repair

1. Incorporation 5’to 3’

2. Proofreading exonuclease 3’to 5’

Bionano – Inorganic-microfab interfaces

• Metal-oxide-semiconductors (sponge silicateins for Ti  & Ga oxides)  • Magnetic components (magnetosomes in magnetotactic bacteria)• Optical fibers & lenses (e.g. venus basket sponge) • Bacterial reduction of salts to metals (e.g. Se, Au, Ag)

• Reading and writing DNA

Reading DNA : Open-source hardware, software, wetware Polonator G007

~10 to $400/Gbp 1E-6 @ >3X redundancy

Synthetic Biology: augmentation & combinatorics (not minimization)

1. Synthetic DNA: 1Mbp per month (Codon Devices)

2. New polymers in vitro – affinity selection (Vanderbilt)

3. Hydrocarbon & other chemical syntheses in E.coli (LS9)

4. Bacterial & stem cell therapies (SynBERC & MGH)

5. New codes: Viral resistant cells & new aminoacids (MIT)

6. Synthetic Ecosystems – Evolve secretion & signaling

7. Interfaces of Genomics & Society

Hierarchical, modular, evolvable

DNA origami -- highly predictable 3D nanostructures

DNA-nanotube-induced alignment of membrane

proteins for NMR structure determination

RothemundNature’06

Douglas, et al. PNAS’07

10 Mbp of DNA / $300 chip

8K Atactic/Xeotron/Invitrogen

Photo-Generated Acid

12K Combimatrix Electrolytic

44K Agilent Ink-jet standard reagents

380K Nimblegen/GA Photolabile 5'protection

Tian et al. Nature. 432:1050 Carr & Jacobson 2004 NAR

Smith & Modrich 1997 PNAS

Spatially patterned chemistry

Amplify pools of 50mers using flanking universal PCR primers &

3 paths to 10X error correction

Mirror world : resistant to enzymes, parasites, predators

Mirror aptamers, ribozymes, etc. require mirror polymerases

352 aminoacid long Dpo4 Sulfolobus DNA polymerase IV347 peptide bonds done; 4 to go.

L-aminoacidsD-nucleotides

(current biosphere)

D-aminoacidsL-nucleotides (Mirror-biopolymers)

• Molecular Biology Central Dogma DNA > RNA > Protein

PCR, T7 RNA pol, in vitro translation.

• Production of devices larger than or toxic to cells.• Directed evolution of drugs & affinity agents.

• Mirror-image proteins

Tony Forster(Vanderbilt)

Duhee Bang (HMS)

Why synthesize (minimal) in vitro self-replication?

113 kbp DNA 151 genes

ideal for comprehensiveatomic, ODE &

stochastic models

Forster & Church

MSB ‘05 GenomeRes.’06Shimizu, Ueda

et al ‘01

Pure in vitro

translating & replicating

system

Genome engineering CAD

70b 15Kb 5Mb 250 Mb

Polymerase in vitro

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

Recombination in vivo E.coli

Error CorrectionMutS 1E-4

Recombination in human cells

Bacterial (Artificial) Chromosomes

BACs

Human(Artificial) Chromosomes

HACs

Sequencing 1E-7

Chemical Synthesis

1E-2

Native DNA computing : Lab Evolution

Reppas/Lin Trp/Tyr exchangeTolonen Ethanol resistance Lenski Citrate utilizationPalsson Glycerol utilizationEdwards Radiation resistanceIngram Lactate productionMarliere ThermotoleranceJ&J Diarylquinoline resistance

(TB)DuPont 1,3-propanediol production

About 3 serial additive changes per 30 days vs 2^30 exhaustive search

rE.coli Strategy #3: ss-Oligonucleotide Repair

Obtain 25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, dam)

Ellis et al. PNAS 2001Constantino & Court. PNAS 2003

DNA Replication Fork

Improved Recombination Frequency:10-4 0.25 (> 3 log increase!)

Multiplex Automated Genome Engineering (MAGE)

Wash with water &

DNA pool (50)

Concentrate, electroporate

Resuspend, bubble, select

O-ring membrane

Concentrate

Wang, Isaacs, Terry

GEMASS Prototype

H. Wang, Church Lab, Harvard, 2008

Recombination-Cycling for Combinatorial Accelerated Evolution

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)

Fraction of mutated sites Time*

11 15 7 7/11 3 days

54 45 23 23/54 9 days

* Continuous cycling

Scaling & Automation Increase Efficiency of Recombination

Wang, Isaacs, Carr, Jacobson, Church

Avogadro scale, >>Yottaflops (from CMOS to sea moss)

Ultra-parallel 1038 units (lab libraries:108 to 1015 25mers)

AdaptableEvolution (years), Immune (days), Neural (seconds)

Thermodynamic limit 2x1019 op/J (irreversible) 3 x1020 for polymerase (1010 for current computers)

Memory density: Neural: (1012 op/s & 106 bits)/mm3, DNA: (103 op/s & 1 bit)/nm3

Error rate: DNA: 10-9 ; RNA/protein: 10-4

Biofuel: 4x107 J/kg (~=$) Adleman 1994

.

Multiplex Automated Genome Engineering (MAGE)

syringe pump

electrically actuated valves

electroporation cuvette w/ membrane filter

OD sensor

data acquisition systemcomputer communication /

Wang, Isaacs, Terry

Fab vs. Bio-fab+ Plays well with digital computers - No habla C++- Doesn’t get DNA + DNA is it’s native digital media- Needs us to replicate + We need them- Needs expensive Fab (e.g. ICs) + Simple or complex inputs - Intelligent Design + Evolution

Cross-feeding symbiotic systems:aphids & Buchnera

• obligate mutualism• nutritional interactions: amino acids & vitamins• established 200-250 million years ago• close relative of E. coli with tiny genome (618~641kb)

Aphids

http://buchnera.gsc.riken.go.jphttp://buchnera.gsc.riken.go.jp

MILKFTWVMILKFTWV HR

Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).

Pink= enzymes apparently missing in Bucherna

trp/tyrA pair of genomes shows best co-growth

Reppas, Lin et al. ; Accurate Multiplex Polony Sequencing

of an Evolved Bacterial Genome 2005 Science

SecondPassage

First Passage

Synthetic genome pair evolution

Co-evolution of mutual biosensors/biosynthesissequenced across time & within each time-point

Independent lines of Trp & Tyr co-culture

5 OmpF: (pore: large,hydrophilic > small)

42R-> G,L,C, 113 D->V, 117 E->A

2 Promoter: (cis-regulator) -12A->C, -35 C->A

5 Lrp: (trans-regulator) 1b, 9b, 8b, IS2 insert, R->L in

DBD.

Heterogeneity within each time-point .

Reppas, Shendure, Porecca -12 -11 -10 -9 -8 -7 -6

At late times Tyr- becomes prototroph!

Reducing costs of open-sourcehardware & wetware

Factor • 30 Equipment speed: from 1 up to 30 Mpixels/sec camera• 4 Equipment cost: from $500K down to $150K (Danaher Inc)• 36 Parallelism: 36 flow-cells per camera, 2 billion beads ------------------• 75 Flow cell volume: 1.5 mm down to 0.0085 mm thin• 40 Kit costs: $2000 down to $50 at standard enzyme costs• 10 Enzymes: $4000/mg down to <$400 (Enzymatics Inc)• 50 Genomic subset (Exome – 1% genome)