Transcript George Church

Fabricating with DNA
George Church Wed 22-Aug-2007 9:15 – 9:30 AM
4th Fab Lab Forum & Digital Fabrication Symposium
Thanks to:
AppliedBiosystems, Helicos, Roche454,
Illumina, CGI, IBS, Affymetrix, Enzymatics
PGP Volunteers & Donors !
Fab vs. Bio-fab
+ Plays well with digital computers
- Doesn’t get DNA
- Needs us to replicate
- Needs expensive Fab (e.g. ICs)
- Intelligent Design
- No habla C++
+ DNA is it’s native digital media
+ We need them
+ Simple or complex inputs
+ 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
Rothemund
Nature’06
Douglas, et al.
PNAS’07
DNA-nanotube-induced
alignment of membrane
proteins for NMR
structure determination
Open-source hardware, software, wetware, ELSware
for DNA reading & writing
78-84
87-97
Church, Gilbert
Genomic Sequencing
(electrotransfer, chemiluminescence, film scanner)
Higgins, Richerich, Auger, Smith Multiplex Sequencing
Polony
A 97-99
B 02-03
Rob
Mitra
Greg
Porecca
MJR slide-cycler
Jay
Shendure
GSI Microarray scanner
Open-source hardware, etc: Personal Genome Project
E07
Faster XYZ
D05 = C03 + fluidics
F07 : $106K
(down from $500K)
including computer
Rich
Terry
G07:
internal autosampler
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. 432:1050
Carr & Jacobson 2004 NAR
Smith & Modrich 1997 PNAS
Synthetic Biology:
augmentation & combinatorics (not minimization)
1.
2.
3.
4.
5.
6.
7.
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
Mirror world:
resistant to enzymes, parasites, predators
Mirror aptamers, ribozymes, etc.
require mirror polymerases
834 AA Thermus Polymerase 834 AA
352 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.
Why synthesize (minimal)
in vitro self-replication?
• 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
Duhee Bang (HMS)
Tony Forster
(Vanderbilt)
Pure
in vitro
translating &
replicating
system
ideal for
comprehensive
atomic, ODE &
stochastic
models
Forster &
Church
MSB ‘05
GenomeRes.’06
Shimizu, Ueda
et al ‘01
113 kbp DNA 151 genes
Genome engineering CAD
Recombination
in vivo E.coli
Polymerase
in vitro
70b
15Kb
Error
Correction
MutS 1E-4
Chemical
Synthesis
1E-2
5Mb
Bacterial (Artificial)
Chromosomes
BACs
Recombination
in human cells
250 Mb
Human(Artificial)
Chromosomes
HACs
Sequencing
1E-7
Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church
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)
Improved Recombination Frequency:
10-4  0.25 (> 3 log increase!)
Multiplex Automated Genome Engineering (MAGE)
Wash with
water &
DNA pool
(50)
Concentrate
O-ring
Concentrate,
electroporate
Wang, Isaacs, Terry
membrane
Resuspend,
bubble,
select
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
Recombination-Cycling UAG to UAA E.coli Essential Genes
Mutation Distribution: 11 oligos, 15 cycles
Mutation Distribution: 54 oligos, 45 cycles
25
Frequency
20
15
10
5
0
0
1
2
3
4
5
6
7
# mutations/clone
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
Palsson
Edwards
Ingram
Stephanopoulos
Marliere
J&J
DuPont
Church
Citrate utilization
Glycerol utilization
Radiation resistance
Lactate production
Ethanol resistance
Thermotolerance
Diarylquinoline resistance (TB)
1,3-propanediol production
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)
MILKFTWV
MILKFTWV HR
Aphids
http://buchnera.gsc.riken.go.jp
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).
Synthetic genome pair evolution
Second
Passage
First
Passage
trp/tyrA pair of genomes shows best cogrowth
Reppas, Lin et al. ; Accurate Multiplex Polony Sequencing
of an Evolved Bacterial Genome 2005 Science
Co-evolution of mutual biosensors/biosynthesis
sequenced 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 .
At late times Tyr- becomes prototroph!
Reppas, Shendure, Porecca
-12 -11 -10 -9
-8 -7
-6
.