George Church Wed 22-Aug-2007 9:15 – 9:30 AM4th Fab Lab Forum & Digital Fabrication Symposium

Thanks to:

Fabricating with DNA

AppliedBiosystems, Helicos, Roche454, Illumina, CGI, IBS, Affymetrix, Enzymatics

PGP Volunteers & Donors !

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

Bio – Inorganic interfaces • Metal-oxide-semiconductors (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)

DNA origami -- highly predictable 3D nanostructures

DNA-nanotube-induced alignment of membrane

proteins for NMR structure determination

RothemundNature’06

Douglas, et al. PNAS’07

Open-source hardware, software, wetware, ELSwarefor DNA reading & writing

A 97-99

Greg Porecca

Rob Mitra

B 02-03 Jay

Shendure

MJR slide-cyclerGSI Microarray scanner

78-84

87-97

Church, Gilbert Genomic Sequencing (electrotransfer, chemiluminescence, film scanner)Higgins, Richerich, Auger, Smith Multiplex Sequencing

Polony

Open-source hardware, etc: Personal Genome Project

D05 = C03 + fluidics

Rich Terry

E07Faster XYZ

F07 : $106K (down from $500K)including computer

G07: internal autosampler

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

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

Mirror world: resistant to enzymes, parasites, predators

Mirror aptamers, ribozymes, etc.

require mirror polymerases

834 AA Thermus Polymerase 834 AA352 AA Dpo4 Sulfolobus DNA polymerase IV 174 AA DNA Polymerase X African Swine Fever Virus.

2004: Bang & Kent: A One-Pot Total Synthesis of Crambin (46-mer)

2006: Torbeev &Kent: A 203-mer Covalent Dimer HIV-1 Protease.

• Molecular biology kits: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

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

Multiplex Automated Genome Engineering (MAGE)

syringe pump

electrically actuated valves

electroporation cuvette w/ membrane filter

OD sensor

data acquisition systemcomputer communication /

Wang, Isaacs, Terry

Recombination-Cycling UAG to UAA E.coli Essential Genes

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

Intelligent Design & Metabolic Evolution

Lenski Citrate utilizationPalsson Glycerol utilizationEdwards Radiation resistanceIngram Lactate productionStephanopoulosEthanol resistance Marliere ThermotoleranceJ&J Diarylquinoline resistance

(TB)DuPont 1,3-propanediol productionChurch Trp/Tyr exchange

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!

.