Transcript l - Church Lab

Synthetic, constructive,
Adaptive Biology
George Church Wed 20-Sep-2006
Science Center Hall E
Thanks to:
NHGRI Seq Tech 2004: Agencourt, 454, Microchip,
2005: Nanofluidics, Network, VisiGen
Affymetrix, Helicos, Solexa-Lynx
iGEM International Genetically
Engineered Machines : 37 teams
Harvard iGEM team 2006
DNA Nanostructures
Tiffany Chan
Katherine Fifer
Valerie Lau
Matthew Meisel
Cyanobacteria
Hetmann Hsieh
Jeffrey Lau
Zhipeng Sun
David Ramos
Cell surface targeting
Perry Tsai
Lewis Hahn
Teaching Fellows
Chris Doucette
Shawn Douglas
Nicholas Stroustrup
Advisors
Pamela 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
Genetic code: UUU encodes Phe = F etc
Stop codons: UAG, UAA, UGA
Inheritance
DNA
Structures
Messenger
Sensors/structures/catalysts
RNA
Protein
Sensors
Inherited epigenetic states
(non-standard view)
iGEM project 1:
Cyanobacterial Kai clock
24 hr cycle
programmable time
(works in vitro with
only 3 proteins)
2. DNA nanobox for controlled
release of protein-pharmaceuticals
iGEM project 3:
Cell surface targeting / bridging
3 Exponential technologies
Computation &
Communication
(bits/sec~m$)
1E+13
1E+11
1E+9
1E+7
1E+5
urea
1E+3
operons
Synthesis
(amu/project~M$)
tRNA
B12
1E+1
1E-1
E.coli
telegraph
1E-3
1830
1850
1870
Analysis
(kamu~base/$) tRNA
1890
1910
1930
1950
1970
1990
2010
Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson
2003 ; Kurzweil 2002; Moore 1965
DOE Biofuel Goals
Miscanthus v Panicum (switchgrass) 22 v 10 tons/ha
Goals: 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.
OFF
ON
ON
Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech.
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
(= 2 E.coli genomes or 20 Mycoplasmas /chip)
Digital Micromirror Array
~1000X lower oligo costs
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
Amplify pools of 50mers using
flanking universal PCR primers and
three paths to 10X error correction
Tian et al. Nature. 432:1050; Carr & Jacobson
2004 NAR; Smith & Modrich 1997 PNAS
‘Next Generation’
Technology Development
Multi-molecule Our role
AB/APG/BEC Seq by Ligation (SbL)
454/Roche
Paired ends, emulsion
SLXA/IVGN/NEB Multiplexing & polonies
CGI
Seq by Ligation (SbL)
Affymetrix
Software
Single molecules
Helicos Biosci
Pacific Biosci
Agilent
Visigen Biotech
SAB, cleavable fluors
Advisor KPCB
Nanopores
AB
Smart therapeutics: Environmentally controlled
invasion of cancer cells by engineered bacteria.
Anderson et al. J Mol Biol. 2006
Regulated Capsule
TonB, DapD
& new genetic code
for safety
Optical imaging: bacteria, viruses, and mammalian cells encoding lightemitting 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
New in vivo genetic code: resistant to all
viruses; novel amino acids
Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR
e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability
TTT
TTC
F
TTA
30362
TCT
11495
TAT
22516
TCC
11720
TAC
18932
TCA
9783
S
21999
TGT
Y
16601
TGC
C
8816
TAA
STOP
2703
TGA
STOP
1256
STOP
326
TGG
W
20683
17613
CGT
28382
13227
CGC
29898
20888
CGA
39188
CGG
7399
24159
AGT
11970
29385
AGC
45687
AGA
14029
AGG
43719
GGT
25918
GGC
4 18602
TCG
12166
TAG
CTT
15002
CCT
9559
CAT
CTC
15077
CCC
7485
CAC
CTA
5314
CCA
11471
CAA
71553
CCG
31515
CAG
41309
ACT
12198
AAT
34178
ACC
31796
AAC
9670
AAA
TTG
CTG
L
L
ATT
ATC
I
ATA
ATG
M
GTT
GTC
GTA
GTG
V
P
T
5967
ACA
37915
ACG
19624
AAG
24858
GCT
20762
GAT
20753
GCC
34695
GAC
14822
GCA
35918
GCG
A
27418
GAA
45741
GAG
H
Q
N
K
D
E
1
53641
GGA
24254
GGG
7048
R
4859
S
3 21862
R
2 1692
Isaacs
Church
Forster
2896
33622
Carr
Jacobson
40285
G
10893
15090
Jahnz
Schultz
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 protein
4. Add selectable gene dependent on above
Schultz tRNAs
http://www.sciencemag.org/cgi/reprint/302/5645/584.pdf
Evolving orthogonal tRNA
charging enzymes
http://schultz.scripps.edu/Images_103105/research_fig3.jpg
Microbial lab evolution
Lenski
Church
Palsson
Edwards
Ingram
Stephanopoulos
Marliere
J&J
DuPont
Citrate utilization
Trp/Tyr exchange
Glycerol utilization
Radiation resistance
Lactate production
Ethanol resistance
Thermotolerance
Diarylquinoline resistance
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)
Internal view
of the aphid.
(by T. Sasaki)
Bacteriocyte
(Photo by T.
Fukatsu)
Aphids
http://buchnera.gsc.riken.go.jp
Buchnera
(Photo by
M. Morioka)
Shigenobu et al. Genome sequence of the endocellular bacterial symbiont
of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).
Synthetic combinatorics & evolution of
7*7* 4.7 Mbp genomes
First
Passage
Second
Passage
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
Sequence monitoring of evolution
(optimize small molecule synthesis/transport)
8
Doubling time (hr)
7
6
5
Q1
Q3
4
Q2-1
Q2-2
3
Sequence trp-
2
EcNR1
1
0
0
10
20
30
40
50
60
70
80
90 100 110 120 130 140 150
# of passages
Reppas, Lin & Church
Why low error rates?
Goal of genotyping & resequencing  Discovery of variants
E.g. cancer somatic mutations ~1E-6 (or lab evolved cells)
Consensus error rate
1E-4
4E-5
Total errors (E.coli)
Bermuda/Hapmap
454 @40X
(Human)
500
600,000
200
240,000
3E-7
Polony-SbL @6X
0
1800
1E-8
Goal for 2006
0
60
Also, effectively reduce (sub)genome target size by enrichment for
exons or common SNPs to reduce cost & # false positives.
Mutation Discovery in Engineered/Evolved E.coli
Position
Type
Gene
Location
ABI
Confirm
Comments
986,334
T>G
ompF
Promoter-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
l-3274
T>C
cI
Glu > Glu

l-red heterogeneity
l-9846
T>C
ORF61
Lys > Gly

l-red heterogeneity
Shendure, Porreca, et al. (2005) Science 309:1728
ompF - non-specific transport channel
Can increase import & export capability simultaneously
AAAGAT
CAAGAT
-12 -11 -10 -9
-8 -7
• Promoter mutation at
position (-12)
• Makes -10 box more
consensus-like
-6
• Glu-117 → Ala (in the pore)
• Charged residue known to affect
pore size and selectivity
Co-evolution of mutual biosensors
sequenced across time & within each time-point
3 independent lines of Trp/Tyr co-culture frozen.
OmpF: 42R-> G, L, C, 113 D->V, 117 E->A
Promoter: -12A->C, -35 C->A
Lrp: 1bp deletion, 9bp deletion, 8bp deletion, IS2
insertion, R->L in DBD.
Heterogeneity within each time-point reflecting
colony heterogeneity.
.