Greedy Algorithms in the Libraries of Biology

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Greedy Algorithms in the Libraries of Biology
17-Apr-2008 330-345 PM Avogadro-Scale
Computing MIT Bartos E15
Thanks to
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Is biology optimal?
Present 26720 km/h 4500m pm-Mm
3oK 2000 yr
Human Past Locomotion 50 km/h Ocean
depth 75m Visible l .4-.7 m Cold 0oC Memor
y 20 yr
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3 Exponential technologies1 to 18 month doubling
times
Computation Communication
Gb chips
human
tRNA
urea
B12
Synthetic chemistry
telegraph
Analytic
tRNA
Shendure J, Mitra R, Varma C, Church GM, 2004.
Carlson 2003 Kurzweil 2002 Moore 1965.
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Avogadro scale, gtgtYottaflops (from CMOS to sea
moss)

• Ultra-parallel 1038 units (lab libraries108 to
  1015 25mers)
• Adaptable
• Evolution (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
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DNA error rates
3. Mismatch repair
DNA Replication Fork
2. Proofreading exonuclease 3to 5
Ellis et al. PNAS 2001 Constantino Court. PNAS
2003
1. Incorporation 5to 3
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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

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Reading DNA Open-source hardware, software,
wetware Polonator G007
10 to 400/Gbp 1E-6 _at_ gt3X redundancy
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Synthetic Biology augmentation combinatorics
(not minimization)

• Synthetic DNA 1Mbp per month (Codon Devices)
• New polymers in vitro affinity selection
  (Vanderbilt)
• Hydrocarbon other chemical syntheses in E.coli
  (LS9)
• Bacterial stem cell therapies (SynBERC MGH)
• New codes Viral resistant cells new aminoacids
  (MIT)
• Synthetic Ecosystems Evolve secretion
  signaling
• Interfaces of Genomics Society

Hierarchical, modular, evolvable
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DNA origami -- highly predictable 3D
nanostructures
Rothemund Nature06 Douglas, et al. PNAS07
DNA-nanotube-induced alignment of
membrane proteins for NMR structure determination
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10 Mbp of DNA / 300 chip
Spatially patterned chemistry

• 8K Atactic/Xeotron/Invitrogen
• Photo-Generated Acid
• 12K Combimatrix Electrolytic
• 44K Agilent Ink-jet standard reagents
• 380K Nimblegen/GA Photolabile 5'protection

Amplify pools of 50mers using flanking universal
PCR primers 3 paths to 10X error correction
Tian et al. Nature. 4321050 Carr Jacobson
2004 NAR Smith Modrich 1997 PNAS
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Mirror world resistant to enzymes,
parasites, predators

• Mirror aptamers, ribozymes, etc. require mirror
  polymerases
• 352 aminoacid long Dpo4 Sulfolobus DNA
  polymerase IV
• 347 peptide bonds done 4 to go.

D-aminoacids L-nucleotides (Mirror-biopolymers)
L-aminoacids D-nucleotides (current biosphere)
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Why synthesize (minimal) in vitro
self-replication?

• Molecular Biology Central Dogma
• DNA gt RNA gt 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)
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Pure in vitro translating replicating system
113 kbp DNA 151 genes
ideal for comprehensive atomic, ODE
stochastic models Forster Church MSB 05
GenomeRes.06Shimizu, Ueda et al 01
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Genome engineering CAD
Recombination in human cells
Recombination in vivo E.coli
Polymerase in vitro
70b 15Kb
5Mb 250 Mb
Error Correction MutS 1E-4
Human(Artificial) Chromosomes HACs
Bacterial (Artificial) Chromosomes BACs
Chemical Synthesis 1E-2
Sequencing 1E-7
Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson,
Church
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Native DNA computing Lab Evolution
About 3 serial additive changes per 30 days
vs 230 exhaustive search
Reppas/Lin Trp/Tyr exchange Tolonen Ethanol
resistance Lenski Citrate utilization Palsson G
lycerol utilization Edwards Radiation
resistance Ingram Lactate production Marliere Th
ermotolerance JJ Diarylquinoline resistance
(TB) DuPont 1,3-propanediol production
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rE.coli Strategy 3 ss-Oligonucleotide Repair
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, dam)
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Multiplex Automated Genome Engineering (MAGE)
Wash with water DNA pool (50)
Concentrate
O-ring
membrane
Resuspend, bubble, select
Concentrate, electroporate
Wang, Isaacs, Terry
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GEMASS Prototype
H. Wang, Church Lab, Harvard, 2008
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Recombination-Cycling for Combinatorial
Accelerated Evolution
Mutation Distribution 11 oligos, 15 cycles
Mutation Distribution 54 oligos, 45 cycles
Continuous cycling

• Scaling Automation
• Increase Efficiency of Recombination

Wang, Isaacs, Carr, Jacobson, Church
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Avogadro scale, gtgtYottaflops (from CMOS to sea
moss)

• Ultra-parallel 1038 units (lab libraries108 to
  1015 25mers)
• Adaptable
• Evolution (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
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.
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Multiplex Automated Genome Engineering (MAGE)
syringe pump
computer communication /
data acquisition system
electrically actuated valves
OD sensor
electroporation cuvette w/ membrane filter
Wang, Isaacs, Terry
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Fab vs. Bio-fab

• Plays well with digital computers - No habla
  C
• - Doesnt get DNA DNA is its native
  digital media
• Needs us to replicate We need them
• Needs expensive Fab (e.g. ICs) Simple or
  complex inputs
• Intelligent Design Evolution

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Cross-feeding symbiotic systemsaphids Buchnera

• obligate mutualism
• nutritional interactions amino acids vitamins
• established 200-250 million years ago
• close relative of E. coli with tiny genome
  (618641kb)

MILKFTWV MILKFTWV HR
Aphids
http//buchnera.gsc.riken.go.jp
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Pink enzymes apparently missing in Bucherna
Shigenobu et al. Genome sequence of the
endocellular bacterial symbiont of aphids
Buchnera sp.APS. Nature 407, 81-86 (2000).
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Synthetic genome pair evolution
Second Passage
First Passage
?trp/?tyrA pair of genomes shows best co-growth
Reppas, Lin et al. Accurate Multiplex Polony
Sequencing of an Evolved Bacterial
Genome 2005 Science
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Co-evolution of mutual biosensors/biosynthesisseq
uenced across time within each time-point

• Independent lines of
• TrpD TyrD co-culture
• 5 OmpF (pore large,hydrophilic gt small)
• 42R-gt G,L,C, 113 D-gtV, 117 E-gtA
• 2 Promoter (cis-regulator)
• -12A-gtC, -35 C-gtA
• 5 Lrp (trans-regulator)
• 1bD, 9bD, 8bD, IS2 insert, R-gtL in DBD.
• Heterogeneity within each time-point .

At late times Tyr- becomes prototroph!
Reppas, Shendure, Porecca
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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 lt400
  (Enzymatics Inc)
• 50 Genomic subset (Exome 1 genome)