Alfonso Jaramillo Presentation

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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