Download - Morten Sommer, MIT/Harvard DOE GtL Center Novozyme 30-Jun-2006 CAD for Synthetic Microbial Biofuels
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Morten Sommer, MIT/Harvard DOE GtL CenterNovozyme 30-Jun-2006
CAD for Synthetic Microbial Biofuels
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Our DOE GtL Center goals & strengths
1. Basic enabling technologies: omics, models,
genome synthesis, evolution, sequencing
2. Fermentative production of alcohols & biodiesel.
3. Improving photosynthetic and conversion efficiencies.
4. Harnessing new insights from ecosystems.
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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
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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
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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
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Programmable ligand-controlled riboregulators to monitor metabolites.
Bayer & Smolke 2005 Nature Biotech.
ON
ON
OFF
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Smart therapeutics example: 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
Metabolic constraintsRegulated Capsule
TonB, DapD& new genetic codes
for safety
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LPS- Capsule+ Dap- for safety
7
DapD
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rE.coli: new in vivo genetic codes
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
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Competition & cooperation
• Cooperation between two auxotrophs– Overall fitness depends on secretion– Over-production, increase of export
• Competition among each sub-population– The fastest growing one wins– Increase of uptake
• Coupling between evolution of import and export properties?– Amplified genes– Transporter & pore genes
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Cross-feeding symbiotic systems:aphids & Buchnera
• obligate mutualism• nutritional interactions: amino acids and vitamine• 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
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Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).
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Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).
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ODE based simulation of population dynamics of cross-feeding ∆Trp-∆TyrQuestions:
• When mixed in minimum medium, how do the cell population and the amino acid concentrations change over time?
• What happens when the strains evolve?– improve on amino acid
imports– improve on amino acid
synthesis and/or exports
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Governing ODE system
density of ∆Trp (gBM/ml)
density of ∆Tyr (gBM/ml)
conc. of Trp (mmol/ml)
conc. of Tyr (mmol/ml)
growth rate constant of ∆Trp ([(mmol/ml Trp)-hr]-1)
growth rate constant of ∆Tyr ([(mmol/ml Tyr)-hr]-1)
Tyr excretion rate constant of ∆Trp (mmol/gBM-hr)
Trp excretion rate constant of ∆Tyr (mmol/gBM-hr)
=0.05 Trp requirement of ∆Trp (mmol/gBM)
=0.13 Tyr requirement of ∆Tyr (mmol/gBM)
Initial conditions:
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density of ∆Trp (gBM/ml)density of ∆Tyr (gBM/ml)conc. of Trp (mmol/ml)conc. of Tyr (mmol/ml)
growth rate constant of ∆Trp ([(mmol/ml
Trp)-hr]-1) growth rate constant of ∆Tyr ([(mmol/ml
Tyr)-hr]-1)
Tyr excretion rate constant of ∆Trp
(mmol/gBM-hr)
Trp excretion rate constant of ∆Tyr
(mmol/gBM-hr)
=0.05 Trp requirement of ∆Trp
(mmol/gBM)
=0.13 Tyr requirement of ∆Tyr
(mmol/gBM)
“Steady-state” solution:
Variables:
Parameters:
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Invasion of advantageous mutants
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Fong SS, Burgard AP, Herring CD, Knight EM, Blattner FR, Maranas CD, Palsson BO. In silico design and adaptive evolution of Escherichia coli for production of lactic acid. Biotechnol Bioeng. 2005 91(5):643-8.
Rozen DE, Schneider D, Lenski RE Long-term experimental evolution in Escherichia coli. XIII. Phylogenetic history of a balanced polymorphism. J Mol Evol. 2005 61(2):171-80
Andries K, et al. (J&J) A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science. 2005 307:223-7.
Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome Science 2005 309:1728 (Select for secretion & ‘altruism’).
Intelligent Design & Metabolic Evolution
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‘Next Generation’ Technology Development
Multi-molecule Our roleAffymetrix Software454 LifeSci Paired ends, emulsionSolexa/Lynx Multiplexing & polonyAB/APG Seq by Ligation (SbL)Complete Genomics SbLGorfinkel Polony to Capillary
Single molecules Helicos Biosci SAB, cleavable fluorsPacific Biosci Advisor KPCBAgilent Nanopores Visigen Biotech AB
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HPLC autosampler
(96 wells)syringe pump
Polony Sequencing EquipmentHMS/AB/APG
microscope
with xyz
controls
flow-cell
temperature
control
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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
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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.
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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
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• 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
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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
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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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Prochlorococcus 40ºN - 40ºS
Ocean chl a (Aug 1997 –Sept 2000)Provided by the SeaWiFS Project, NASA
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-Glc-1P ADP-Glc -1,4-glucosyl-glucan glycogenCentralCarbonMetabol.
glgC
glgX
glgA glgB
glgP
Glycogen metabolism
Time (hours)
0 4 8 12 16 20 24 28 32 36 40 44 48
Nor
mal
ized
Exp
ress
ion
0.1
1
10
glgAglgBglgCglgXglgP
Zinser et al. unpublZinser et al. unpubl..
Light regulated Prochlorococcus metabolism
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Photosynthetic Genes in Phage
Podovirus P-SSP7 46 kb
PC HLIPs Fd D1
12kb 24kb
PC HLIPs Fd D1
12kb 24kb
~500 bp
HLIPs D1 D2
6.4kb 2.8kb
~500 bp
Myovirus P-SSM4 181 kbHLIPs D1 D2
6.4kb 2.8kb
Lindell, Sullivan, Chisholm et al. 2004Lindell, Sullivan, Chisholm et al. 2004
HLIP D1
Myovirus P-SSM2 255 kb
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RNA Responses to Phage
MED4-0682 (60 aa Conserved URF)
Phage SSP7 psbA
MED4 host psbA
Lindell,Lindell, Sullivan, Zinser, ChisholmSullivan, Zinser, Chisholm
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Our DOE GtL Center goals & strengths
1. Basic enabling technologies: omics, models,
genome synthesis, evolution, sequencing
2. Fermentative production of alcohols & biodiesel.
3. Improving photosynthetic and conversion efficiencies.
4. Harnessing new insights from ecosystems.
![Page 32: Morten Sommer, MIT/Harvard DOE GtL Center Novozyme 30-Jun-2006 CAD for Synthetic Microbial Biofuels](https://reader030.vdocuments.us/reader030/viewer/2022032800/56649d435503460f94a1fee1/html5/thumbnails/32.jpg)
Morten Sommer, MIT/Harvard DOE GtL CenterNovozyme 30-Jun-2006
CAD for Synthetic Microbial Biofuels
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.