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Synthetic Biology
Alfonso JARAMILLO
Leader Synth-Bio group
Epigenomics Prg., Genopole-CNRS
Ass. Prof. Ecole Polytechnique
http://synth-bio.org
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Outline of Talk
Synthetic Biology Example: Artemisin story Design principles in SB Introduction to biological devices
Outline
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Synthetic Biology
Making biology more engineerable / Engineer complex things
Synthetic biology extends the spirit of genetic engineering tofocus on de novoprotein and RNA, on whole systems of genes
or gene products. This is opposed to introducing individual
mutations, genes or pathways in previous works.
Systems biology provides knowledge how parts of the celloperate together - Synthetic Biology (SB) provides a true
engineering approach to tailor sub-cellular biology as a system
of interacting modules
New tools available such as computer models andbioinformatics, rapid synthesis, better experimental techniquesto explore gene interactions
Industry will benefit from its tremendous potential and impact(materials synthesis, energy production, sensing, )
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Enabling breakthroughs in a postgenomic era
Advances in computing power Internet Genomic sequencing Crystal structures of proteins High through-put technologies
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Availability of DNA synthesis
For example the bacteriaMycoplasma
genitalium has the smallest genome out of all
living cells: 517 genes over 580 kb.
Minimal costs of oligo creation (not including
error-checking):
Mid 1990s: $1/bp = $580,000
Circa 2000: $0.35/bp = $203,000
2006: $0.11/bp = $63,800
Ambitious prediction of not-too-distant
future (Church et al, 2004): $0.00005/bp =
$29
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Engineering new biological systems
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Path 1: the construction of engineered
DNA, which allows manipulation at every
level of the natural hierarchy.Path 2: the use of engineered DNA toproduce novel nanostructures.
Path 3: the development of nonstandard
amino acids and base pairs, which can thenbe assembled into foldamers and DNA
analogs.
Path 4: the creation of alternative geneticsystems.
Path 5: producing minimal genomes(synthetic chromosomes) and transplanting
them into prokaryotic hosts.Path 6: adding new functions to living
organisms by manipulating cell machinery.
Path 7: the fusion of proteins to produceassemblies with novel functions.
Path 8: the use of peptide synthesis tocreate programmable building blocks that
can assemble further into functional protein
components.
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Example
Microbial production of anti-Malaria drug
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1-3 millionpeople die of malariaevery year
90% are children
300-500 millionpeople infected Source: Roll Back MalariaWorld Malaria Report 2005
Synthetic Biology Against Malaria
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The current cost for an artemisinin-based drug isapproximately $2.50.
Artemisinin generally adds $1.00-1.50 to thecost for drugs
Most developing countries spend less than $4/person/year on health care
As many as 10-12 treatments are needed for eachperson annually
World Health Organization estimates that 700tons will be needed annually
Artemisinin-based drugs
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A chemical synthesis route to artemisinin
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Reduce the cost of artemisinin-based anti-malarial drugs by an order of magnitude.
Approach
Engineer a microorganism to
produce artemisinin from an
inexpensive, renewableresource.
Goal
J. Keasling lab
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Monoterpenes
Sesquiterpenes
Diterpenes
Gene resynthesis improves
amorphadiene production
Pathways for isoprenoid precursor biosynthesis
Amorphadiene
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Monoterpenes
Sesquiterpenes
Diterpenes
Pathways for isoprenoid precursor biosynthesis
Amplified expression of E.
colis native genes that producefarnesyl diphosphate
Amorphadiene
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Monoterpenes
Sesquiterpenes
Diterpenes
Pathways for isoprenoid precursor biosynthesis
Amplified expression of E.
colis native genes that producefarnesyl diphosphate
Amorphadiene
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Yeast
G3P
PEP
PYR
cCoA
CIT
IPP DMAPP
GPP
MEV
Mevalonate pathway
DX
P
DXP pathway
FPPTCACycle
Amorphadiene
Recruiting the mevalonate pathway from yeast
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Acetyl-CoAP
HMGS tHMGRatoBMevT
Mevalonate
FPP
MBISP
PMK MPDMK idi
Mevalonate
ispA
Construction of synthetic mevalonate pathway operons
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Proposed artemisinin biosynthetic pathway
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p450
Amorphadiene Artemisinic Acid
1 2 3
>25 g/L
Current titer
in E. coli
(lab scale)> 1 g/L
P450/AMO Catalyzes 3 Separate Oxidations
Completing the biosynthetic pathway inE. coli
E. coli has been engineered to produce amorphadiene at yields in excess of 0.5 g/L.S. cerevisiae has been engineered to produce amorphadiene at yields of approx 0.1 g/L.A cytochrome P450 and its redox partners can oxidize amorphadiene to artemisinic acid.S. cerevisiae expressing amorphadiene oxidase produces artemisinic acid, which is secretedfrom the cells.
E. coli can functionally express cytochrome P450s that oxidize terpenes.
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E. coli has been engineered to produce amorphadiene at yields inexcess of 0.5 g/L.
S. cerevisiae has been engineered to produce amorphadiene atyields of approximately 0.1 g/L.
A cytochrome P450 and its redox partners can oxidizeamorphadiene to artemisinic acid.
S. cerevisiae expressing amorphadiene oxidase producesartemisinic acid, which is secreted from the cells.
E. coli can functionally express cytochrome P450s that oxidizeterpenes.
Artemisin project
J. Keasling lab
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Design Principles of SB
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Q: But if we dont fully understand all the rules ofbiology, how can we create anything more than basicsystems?
A: We can press our limits by modularizing andsimplifying as much as possible
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Decoupling Design & Fabrication Rules insulating design process from details of fabrication Enable parts, device, and system designers to work together VLSI electronics (1970s)
Abstraction Insulate relevant characteristics from overwhelming detail Simple components that can be used in combination From Physics to Electrical Engineering (1900s)
Standardization of Components Predictable performance Off-the-shelf Mechanical Engineering (1800s) & the manufacturing revolution (e.g. Henry
Ford)
Design principles
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Biological modules for engineering
Chassis: Bacterial strain that will receive the engineeredsystems.
Parts: Fragment of DNA with a given functionality. Devices: Assembly of parts with a given functionality and
given interface. Specifications I/O is given by proteins and signals
Systems: Assembly of devices with a given functionality I/O is given only by signals
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I need a few DNAbinding proteins.
Heres a set of DNA bindingproteins, 1N, that eachrecognize a unique cognateDNA site, choose any.
Get me this DNA.
Heres your DNA.
Can I havethree inverters?
Heres a set of PDP
inverters, 1N, that eachsend and receive via afungible signal carrier, PoPS.
TAATACGACTCACTATAGGGAGA DNA
Zif268, Paveltich & Pabo c. 1991
Parts
PoPSNOT.1
PoPS PoPS Devices
PoPS
NOT.2
PoPS
NOT.3
PoPS
NOT.1
Systems
Abstraction hierarchy
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Modularity by design
DeviceOff-the-shelf
partsDesignIdea
Building a radio with off-the-shelf parts
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I13453 B0034 I15008 B0034 I15009 B0015tetR
R0040 B0034 I15010 B0015
BBa_M30109 =
Notice that for the MIT registry, anycombination of parts (e.g. devicesand systems) is a part.
Off-the-shelf biological parts and devices
Promoter RBS CDS Terminator Tag Primer Operator
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27S PXE
Biobricks
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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Registry of Standard Biological Parts
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iGEM competition
http://parts.mit.edu/igem07/index.php/Paris
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Biological devices
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Devices
They should be able to always produce the same ouput from thesame input.
Need of specification of transfer functions and I/O proteins/molecules. The engineer will be able to model the devices from the specifications
without needing to know the internals. Encapsulation of data.
Devices with interface with each other. Need of a standard. In the real world the devices will interact with the chassis.
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LacI CI inverter
CILacI
Devices
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cI-857OLac RBS T
CILacI
LacI
CI
Devices
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Inverter.2 Inverter.3Inverter.1
Systems
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Device-Level System Diagram
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Parts- and Device-Level System Diagram
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DNA Layout
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cI-857OLac RBS T
cILacI
cI-857RBS T
cI
O
PoPSin
PoPSout
LacI
cIPoPSout
PoPSin
Device Interface
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cIRBST
O
cI
PoPSIN
cIRBST
O
PoPSOUT
Polymerase Per Second = PoPS!
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cIRBST
O
PoPSOUT
PoPSIN
cI
PoPSOUTPoPSIN
INVERTER
PoPSOUTPoPSINPoPSOUT
PoPS Source (Any)
Polymerase Per Second = PoPS!
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Part characterisation
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From biological discovery to an engineered device
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The device is re-engineered usingstandardised biological parts
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Multi-scale computational design for SB
De novo design of proteins:
DESIGNER PROTDES
De novo design of:
Transcriptional networks GENETDES ASMPARTS
RNA networks RNADES
De novo design of metabolicpathways by retro-biosynthesis DESHARKY
Network inference frommicroarray & proteomics resp. INFERGENE
Macromolecules
Biological networks
Cellular systems
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Modelling & Characterisation
Characterization biological part models (Asmparts)
Construction of a computational
promoter library
Combinatorial promoter
Systematic characterisation and modelling of biological
systems for their re-use.
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In silico genome evolution and design
Evolution moves:
Add/remove TF or enzyme Replace promoter Add/remove operon Modify kinetic parameters
Biological part models (Asmparts) Desharky to move in metabolic space Fitness/scoring function:
Use chassis model to estimate cell growth Cost/benefit model:
Expressing genes is decremental to growth Expressing useful pathways contributes to
growth
FBA for fast metabolic reactions, ODEsfor slow transcriptional ones.
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Conclusions
Synthetic biology aims at the engineering of biological systemswith targeted behaviour
This requires making biology more engineerable: Abstraction, modules, standardisation De novoprotein and RNA, on whole systems of genes or gene products
requires new procedures.
New tools available computer models and bioinformatics, rapid synthesis, better
experimental techniques to explore gene interactions
Industry will benefit from its tremendous potential and impact(materials synthesis, energy production, sensing, )
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Abstraction-Parts-Devices-Systems
SpecificationModularity
SimulationOptimization
H2 production
Oxygen consumption
Regulation
BioModularH2: biohydrogen production
Adapted from KEGG
Hydrogen is considered the energy carrierof the future
Use cyanobacteria for photoproduction ofhydrogen:
Photosynthesis produces oxygen Oxygen inhibits hydrogen production by
hydrogenase!
http://biomodularh2.org
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Genome engineering
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Creating a linear genome
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Merging genomes
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Refactoring genomes
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Forster & Church
Oligos for
150 & 776 syntheticgenes(forE.coliminigenome & M.mobile
whole genome respectively)
De novo engineering a cell from known parts