what is synthetic biology? - washington university...

What is Synthetic Biology?

S 5 POSITIVE S 5 NEGATIVE

Words/Phrases that you think of when you hear “SYNTHETIC BIOLOGY”

What’s in a name?News Feature, Nature Biotech, December 2009

What is Synthetic Biology?

S One possible definition: “Synthetic biology is the discovery, invention, and manufacture of biochemical elements and systems to produce tools, materials, organisms, and devices that meet human needs.”

S Two AreasS A) the design and construction of new biological parts, devices, and

systems, andS B) the re-design of existing, natural biological systems for useful purposes.

http://en.wikipedia.org/wiki/File:Wohler_synthesis.gif

Wöhler Synthesis of Urea: The Birth of Synthetic Organic Chemistry (1828)

• First example of combining inorganic reactants (isocyanic acid + ammonia) to produce an organic compound (urea)

• Wöhler to his mentor Berzelius:"I cannot, so to say, hold my chemical water and must tell you that I can make urea without thereby needing to have kidneys, or anyhow, an animal, be it human or dog".

• “We have got to the point in human history where we simply do not have to accept what nature has given us…”

• “By combining elements of engineering, chemistry, computer science, and molecular biology, synthetic biology seeks to assemble the biological tools necessary to redesign the living world…”

• “Synthetic biology will create cheap drugs, clean fuels, and new organisms to siphon carbon dioxide from the atmosphere.”

http://www.newyorker.com/

Why should you be interested in synthetic biology?

What Can We GET From Engineering Biological Systems?

http://doegenomestolife.org/

Toxic Waste

Global Warming

Energy

New Therapeutics

What do we NEED from engineered biological systems and how do we get

there?

Engineered systems must be– Reliable– Predictable– Functionally robust

• Do we need new biology?

• Do we need a new way of engineering?

A Synthetic Biologists Specific Aims:

• Describe and predict the behavior of biological networks– Isolate modules of gene networks (autoregulatory networks, toggle switches, etc.)– Develop theoretical models of gene regulation to predict and describe the behavior

of simple networks– Modeling may be utilized to guide experimentation– Couple well-characterized modules in order to deduce the behavior of large-scale

networks

• Design new genetic components that can serve as modular, global components

– Sensors of environmental stimuli– Probes to explore the behavior of biological networks– Regulators to control genetic, regulatory or metabolic networks

• Recode & engineer whole organisms– rE.coli

Gain a better understanding of gene expression & cellular behavior

Utilize this knowledge to engineer cellular function

Cellular Network: Exhibit remarkably robust, precise behaviorin the absence of our understanding

Cellular Phone: Designed and built by engineers EVERY component is characterized

reduce the complexity of networks from natural complex biological setting to isolate and study modular components that perform a specific function

Modular Cell Biology

Modules: composed of many types of molecules - DNA, RNA, proteins, small molecules - which have discrete functions that arise from interactions among their components

Hartwell, Hopfield, Leibler, Murray Nature 402 (1999)Arnone & Davidson Development 124 (1997)

Biological Complexity

“The sequence provides the framework upon which all the genetics, biochemistryphysiology, and ultimately phenotype depend...The sequence is only the first levelof understanding the genome. All genes and control elements must be identified;their functions in concert as well as in isolation, defined; their sequence variation

worldwide described; and the relation between genome variation and specific phenotypic characteristics determined. Now we know what we have to explain.”

J.C. Venter et al. Science 291 (2001)

Genome Sequences: “the framework”

Systems Biology Synthetic Biology

DNA Sequencing Technologies

Merging Genomes

Essential GenesReduced GenomesEmergent Properties of Reduced-Genome Escherichia coli

Refactoring bacteriophage T7Chan, Kosuri and Endy Molecular Systems Biology (2005)

Genome Restructuring

Hutchison, Venter et al.Science (1999)

Itaya, Fujita et al.PNAS (2006)

Pósfai, Blattner et al. Science (2006)

GenomeTransplantationLartigue, Venter et al.

Science (2007)

GenomeSynthesis

Gibson, Smith et al.

Science (2008)

Lartigue, Glass et al.

Science (2009)

Natural & Synthetic Gene Circuits ‡ Engineering Biology

Sprinzak & Elowitz Nature 438 (2005)

The Design Cycle: Coupling Theory and Experiment (Street and Mayo 1999)

Computational Methods predict the “Best” theoretical solutions

Experimental Methods measure, compare and quantify

success and failure of predictions

PARAMETERIZATION

PREDICTION

Synthetic Gene Networks:• Construction of small gene networks from well-characterized biological parts,

guided by models

Bistable Toggle SwitchGardner, Cantor & Collins Nature 403 (2000)

RepressilatorElowitz & Leibler Nature 403 (2000)

Good Review: Sprinzak & Elowitz Nature 438 (2005)

Freeman Nature 313 (2000)Becskei et al Nature 405 (2000)& EMBO J 20 (2001)Isaacs et al PNAS 100 (2003)

Feedback Loops

Building blocks: Promoters, Repressors and Feedback

Natural Negative Feedback: Promoter (PR) expresses Protein (Cro) which represses PR

Synthetic Negative Feedback: Tighter distribution of protein product than unregulated protein production

Hasty & Collins Nature 420 (2002)

Building blocks: Promoters, Repressors and Feedback

Natural positive feedback: Mos-MEK-p42 MAPK cascade required for Xenopus oocytematuration

Synthetic positive feedback: can be self-perpetuating and essentially irreversible

Hasty & Collins Nature 420 (2002)Ferrell Curr Opin Chem Bio (2002)

Bistability: Double-negative or Positive Feedback Loops

• Bistability: two stable states (minima) are separated by a peak (maximum)

Ferrell Curr Opin Cell Bio (2002)

http://upload.wikimedia.org/wikipedia/commons/5/54/Bistability.svghttp://upload.wikimedia.org/wikipedia/commons/5/54/Bistability.svg

Bistable Toggle SwitchGardner, Cantor & Collins Nature 403 (2000)

Natural toggle-switch: l-phage lysis or lysogeny decision

Synthetic toggle-switch:• “Repress my repressor” cascade• Only requires pulse of one trigger to switch the state

• IPTG (inducer) inhibits lacIrepression of Ptrc-2:

• CI expressed, represses lacI• GFP expressed indefinitely

• Heat (inducer) inhibits cItsrepression of PLs1con:

• lacI expressed, represses cIts• GFP repressed indefinitely

GFP

Flu

ores

cenc

e

Genetic Logic Gates

Hasty & Collins Nature 420 (2002)

The RepressilatorElowitz & Leibler Nature 403 (2000)

Simple computational model of transcriptional regulation with parameters for:• dependence of transcription rate on repressor concentration,• translation rate, • decay rates of the protein and messenger RNA.

Two types of solutions are possible: • system may converge to stable steady state, or• steady state may become unstable, leading to sustained limit-cycle oscillations

Oscillations predicted when:• strong promoters coupled to efficient ribosome-binding sites• tight transcriptional repression (low `leakiness'),• cooperative repression characteristics,• comparable protein and mRNA decay rates

The RepressilatorElowitz & Leibler Nature 403 (2000)

Even well-characterized components

did not yield a well-behaved system

Can we design a biological function from the ground up?

Time (min)

Fluo

resc

ence

Instability Variability

UnpredictableElowitz & Leibler Nature 403 (2000)

Design of cell-cell communication: Genetic Band-Pass Detector

Basu, Gerchman, Collins, Arnold & Weiss Nature 434 (2005)

The PARTS:• LuxI: Enzyme produces AHL• AHL: Signaling molecule, activates LuxR• LuxR: Senses AHL, activates LacIm1 & CI• LacIm1: Weak repressor of GFP• CI: Strong repressor of LacI• LacI: Repressor of GFP

Design of cell-cell communication: Genetic Band-Pass Detector

Basu, Gerchman, Collins, Arnold & Weiss Nature 434 (2005)

BAND-PASS DETECTOR FEATURES:• Completely dependent on strength of LuxR• CI better repressor than LacIm1, better response to LuxR• High-Detect: Upper threshold of AHL for GFP inhibition• Low-Detect: Lower threshold of AHL for GFP inhibition• Band-detect: Middle range of AHL for GFP expression

Changing LuxR strength shifts both the high detect and low detect thresholds for AHL detection and hence GFP expression

Coordinating cellular networks to achieve higher-order patterning

RED: Strong LuxRGREEN: Weak LuxR

[AHL] µ 1 .LuxR

Design of global cell-to-cell communications to generate synchronized oscillations

Danino, Mondragon-Palomina, Tsimring & Hasty Nature 463 (2010)

THE PARTS:• luxI: enzyme produces AHL• AHL: signaling molecule, activates LuxR ABOVE A CONCENTRATION THRESHOLD (Quorum)• LuxR: transcriptional activator of luxI, GFP, & aiiA• aiiA: degrades AHL



Generating synchronized waves of fluorescence in a longer device



3D device shows radial waves after quorum is setup

From circuits to systems: where do we stand?

The total number of systems (circuits) described has increased, but not the complexity of individual systems.

Harnessing biology for next generation biofuels

Image by Eric Steen, JBEI

Image by Jonathan Remis, JBEI

• US consumes 9 million barrels (1,400,000 m3) petroleum / day as motor fuel

Biosynthesis of Artemisinin: A global health success story

Malaria:

300-500 million new infections each year

1-3 million deaths

Will reduce cost of treatment 10-

fold

Artemisinin

Engineering an 11-enzyme Pathway in Parts

Keasling JACS Chem Biol 3 (2007)

Compartmentalization and Shutting off Competing Pathways

Martin et al. Nature Biotech 21 (2003)

Keasling JACS Chem Biol 3 (2007)Pfleger et al Nature Biotech 24 (2006)

Combinatorial Tuning of Intergenic Regions in Synthetic Operon

Occluded RBS

Synthetic scaffolds for selecting optimal operon stoichiometry

Keasling JACS Chem Biol 3 (2007)Dueber et al Nature Biotech 27 (2009)

De novo gene synthesis of tobacco ADS enzyme with E. coli codon optimization

Overlapping Oligo Assembly: DETAILS IN JIM’s LECTURE

Martin et al. Nature Biotech 21 (2003) Keasling JACS Chem Biol 3 (2007)

Functional genomic discovery of native A. annua oxidase

• Create leaf cDNA library from A. annua

• Degenerate PCR from cDNA using lettuce and sunflower (closely related Asteraceae crops that produce terpenes) P450 sequences

• Subclone novel P450 and redox partner into S. cerevisiae

• MIRACLE (three steps catalyzed!) to produce artemisinic acid

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Phylogenetic tree of new A. annua P450

Biosynthesis of Artemisinin: A global health success story

Malaria:

300-500 million new infections each year

1-3 million deaths

Will reduce cost of treatment 10-

fold

Artemisinin

Can we move to a new paradigm?

• Conceive a desired biological function

• Design an engineered biological system

• Build it• It works as predicted

What is Synthetic Biology?What’s in a name?�News Feature, Nature Biotech, December 2009What is Synthetic Biology?Wöhler Synthesis of Urea: The Birth of Synthetic Organic Chemistry (1828)Why should you be interested in synthetic biology?What Can We GET From Engineering Biological Systems?What do we NEED from engineered biological systems and how do we get there?A Synthetic Biologists Specific Aims:Cellular Phone: Designed and built by engineers �EVERY component is characterized�Biological ComplexityGenome Sequences: “the framework”Slide Number 12Slide Number 13Natural & Synthetic Gene Circuits Engineering BiologyThe Design Cycle: Coupling Theory and Experiment (Street and Mayo 1999)Synthetic Gene Networks:Building blocks: Promoters, Repressors and FeedbackBuilding blocks: Promoters, Repressors and FeedbackBistability: Double-negative or Positive Feedback LoopsBistable Toggle Switch�Gardner, Cantor & Collins Nature 403 (2000)Genetic Logic GatesThe Repressilator�Elowitz & Leibler Nature 403 (2000)The Repressilator�Elowitz & Leibler Nature 403 (2000)Can we design a biological function �from the ground up?Design of cell-cell communication: �Genetic Band-Pass Detector�Basu, Gerchman, Collins, Arnold & Weiss Nature 434 (2005)Design of cell-cell communication: �Genetic Band-Pass Detector�Basu, Gerchman, Collins, Arnold & Weiss Nature 434 (2005)Coordinating cellular networks to achieve higher-order patterningDesign of global cell-to-cell communications to generate synchronized oscillations�Danino, Mondragon-Palomina, Tsimring & Hasty Nature 463 (2010)Slide Number 29Slide Number 30Slide Number 31From circuits to systems: where do we stand?Harnessing biology for next generation biofuelsBiosynthesis of Artemisinin: �A global health success storyEngineering an 11-enzyme Pathway in PartsCompartmentalization and �Shutting off Competing PathwaysCombinatorial Tuning of Intergenic Regions in Synthetic OperonSynthetic scaffolds for selecting optimal operon stoichiometryDe novo gene synthesis of tobacco ADS enzyme with E. coli codon optimizationFunctional genomic discovery of native A. annua oxidaseBiosynthesis of Artemisinin: �A global health success storyCan we move to a new paradigm?

what is synthetic biology? - washington university...

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