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George Church Wed 20-Sep-2006 Science Center Hall E
Thanks to:
Synthetic, constructive,
Adaptive Biology
NHGRI Seq Tech 2004: Agencourt, 454, Microchip, 2005: Nanofluidics, Network, VisiGen Affymetrix, Helicos, Solexa-Lynx
Harvard iGEM team 2006DNA NanostructuresTiffany Chan Katherine Fifer Valerie Lau Matthew Meisel CyanobacteriaHetmann Hsieh Jeffrey Lau Zhipeng Sun David Ramos Cell surface targetingPerry Tsai Lewis Hahn
Teaching FellowsChris Doucette Shawn Douglas Nicholas Stroustrup AdvisorsPamela Silver George Church Radhika Nagpal Jagesh V. Shah William Shih Alain Viel
http://openwetware.org/wiki/IGEM:Harvard/2006
The Central Dogma of Molecular Biology
Inheritance Messenger Sensors/structures/catalysts
Structures Sensors Inherited epigenetic states
(non-standard view)
DNA RNA Protein
Genetic code: UUU encodes Phe = F etcStop codons: UAG, UAA, UGA
iGEM project 1: Cyanobacterial Kai clock
24 hr cycle programmable time(works in vitro with
only 3 proteins)
3 Exponential technologies
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~m$)
Synthesis (amu/project~M$)
Analysis(kamu~base/$) tRNA
DOE Biofuel Goals
Miscanthus v Panicum (switchgrass) 22 v 10 tons/haGoals: 2kg Hybrid seeds v 2 tons rhizomes
self-destruction to aid crop rotation, pretreatment$0.10/L goal (NEB >4, corn-EtOH:1.3 soy-diesel:1.93)
Integrated cellulases & fermentation to ethanol, butanol, biodiesel, alkanes $0.02/L
via metabolic engineering & lab evolution
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
Programmable ligand-controlled riboregulators to monitor metabolites.
Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech.
ON
ON
OFF
Genome & Metabolome Computer Aided Design (CAD)
4.7 Mbp new genetic codes new amino acids 7*7 * 4.7 Mbp mini-ecosystems biosensors, bioenergy, high secretors, DNA & metabolic isolation
•Top Design Utility, safety & scalability
CAD-PAM Synthesis (chip & error correction)
Combinatorics Evolution Sequence
How? 10 Mbp of oligos / $1000 chip
8K Atactic/Xeotron/Invitrogen
Photo-Generated Acid
Sheng , Zhou, Gulari, Gao (Houston)
12K Combimatrix Electrolytic
44K Agilent Ink-jet standard reagents
380K Nimblegen Photolabile 5'protection
Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997
PNAS
~1000X lower oligo costs
(= 2 E.coli genomes or 20 Mycoplasmas /chip)
Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction
Digital Micromirror Array
‘Next Generation’ Technology Development
Multi-molecule Our roleAB/APG/BEC Seq by Ligation (SbL)454/Roche Paired ends, emulsionSLXA/IVGN/NEB Multiplexing & poloniesCGI Seq by Ligation (SbL)Affymetrix Software
Single molecules Helicos Biosci SAB, cleavable fluorsPacific Biosci Advisor KPCBAgilent Nanopores Visigen Biotech AB
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
New in vivo genetic code: resistant to all viruses; novel amino acids
TTT
F
30362 TCT
S
11495 TAT
Y
21999 TGT
C
7048
TTC 22516 TCC 11720 TAC 16601 TGC 8816
TTA
L
18932 TCA 9783 TAASTOP
STOP
2703 TGA STOP 1256
TTG 18602 TCG 12166 TAG 326 TGG W 20683
CTT
L
15002 CCT
P
9559 CAT
H
17613 CGT
R
28382
CTC 15077 CCC 7485 CAC 13227 CGC 29898
CTA 5314 CCA 11471 CAA
Q
20888 CGA 4859
CTG 71553 CCG 31515 CAG 39188 CGG 7399
ATT
I
41309 ACT
T
12198 AAT
N
24159 AGT
S
11970
ATC 34178 ACC 31796 AAC 29385 AGC 21862
ATA 5967 ACA 9670 AAA
K
45687 AGA
R
2896
ATG M 37915 ACG 19624 AAG 14029 AGG 1692
GTT
V
24858 GCT
A
20762 GAT
D
43719 GGT
G
33622
GTC 20753 GCC 34695 GAC 25918 GGC 40285
GTA 14822 GCA 27418 GAA
E
53641 GGA 10893
GTG 35918 GCG 45741 GAG 24254 GGG 15090
Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGRe.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability
IsaacsChurch
Forster
CarrJacobson
JahnzSchultz
1
2
3
4
To change the genetic code
1. Free up 1 or more codons by change 30 to 30,000 codons throughout the genome
2. Remove the RF or tRNA dedicated to those codon(s)3. Add orthogonal tRNA and synthetase protein4. Add selectable gene dependent on above
Evolving orthogonal tRNA charging enzymes
http://schultz.scripps.edu/Images_103105/research_fig3.jpg
Microbial lab evolution
Lenski Citrate utilizationChurch Trp/Tyr exchangePalsson Glycerol utilizationEdwards Radiation resistanceIngram Lactate productionStephanopoulosEthanol resistanceMarliere ThermotoleranceJ&J Diarylquinoline
resistanceDuPont 1,3-propanediol production
Cross-feeding symbiotic systems:aphids & Buchnera
• obligate mutualism• nutritional interactions: amino acids and vitamins• established 200-250 million years ago• close relative of E. coli with tiny genome (618~641kb)
Aphids
Internal view of the aphid. (by T. Sasaki)
Bacteriocyte (Photo by T. Fukatsu)
Buchnera (Photo by M. Morioka)
http://buchnera.gsc.riken.go.jphttp://buchnera.gsc.riken.go.jp
Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).
trp/tyrA pair of genomes shows the best co-growth
Reppas, Lin & Church ; Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome(2005) Science 309:1728
SecondPassage
First Passage
Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
# of passages
Do
ub
lin
g t
ime
(h
r)
Q1
Q3
Q2-1
Q2-2
EcNR1
Sequence monitoring of evolution(optimize small molecule synthesis/transport)
Sequence trp-
Reppas, Lin & Church
Consensus error rate Total errors (E.coli)
(Human)
1E-4 Bermuda/Hapmap 500
600,000
4E-5 454 @40X 200 240,000
3E-7 Polony-SbL @6X 0 1800
1E-8 Goal for 2006 0 60
Goal of genotyping & resequencing Discovery of variantsE.g. cancer somatic mutations ~1E-6 (or lab evolved cells)
Why low error rates?
Also, effectively reduce (sub)genome target size by enrichment for exons or common SNPs to reduce cost & # false positives.
Position Type Gene LocationABI
ConfirmComments
986,334 T > G ompFPromoter-
10 Only in evolved strain
985,797 T > G ompF Glu > Ala Only in evolved strain
931,960 ▲8 bp lrp frameshift Only in evolved strain
3,957,960 C > T ppiC 5' UTR MG1655 heterogeneity
-3274 T > C cI Glu > Glu red heterogeneity
-9846 T > CORF6
1Lys > Gly red heterogeneity
Mutation Discovery in Engineered/Evolved E.coli
Shendure, Porreca, et al. (2005) Science 309:1728
• Glu-117 → Ala (in the pore)
• Charged residue known to affect pore size and selectivity
• Promoter mutation at position (-12)
• Makes -10 box more consensus-like
-12 -11 -10 -9 -8 -7 -6
AAAGAT
CAAGAT
Can increase import & export capability simultaneously
ompF - non-specific transport channel
3 independent lines of Trp/Tyr co-culture frozen.
OmpF: 42R-> G, L, C, 113 D->V, 117 E->APromoter: -12A->C, -35 C->ALrp: 1bp deletion, 9bp deletion, 8bp
deletion, IS2 insertion, R->L in DBD.
Heterogeneity within each time-point reflecting colony heterogeneity.
Co-evolution of mutual biosensorssequenced across time & within each time-point
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