Fundamentals of Synthetic Biology

Introduction

Definition The “abstraction hierarchy” Orienting Examples Basic Procures

Basic Manufacture Parts: proteins/etc. Devices: Scaffolds and more Systems: Therapeutic Bacteria Characterization Future of Manufacture Future Applications

Introduction

Definition The “abstraction hierarchy” Orienting Examples Basic Procures

Synthetic BiologyAn engineering science to develop the standards, abstractions, characterization protocols, and parts lists to make genetic engineering of new, complex, biological functions cheaper, faster, scalable and reliable.

NSF ERChttp://www.synberc.org (Berkeley, Harvard, MIT, Prairie View, UCSF)

Review of Central Dogma

The Rosetta Stone

In the reign of the young one who has succeeded his father in the kingship, lord of diadems, most glorious, who has established Egypt and is pious towards the gods, triumphant over his enemies, who has restored the civilized life of men, lord of the Thirty Years Festivals, even as Ptah the Great, a king like Ra, great king of the Upper and Lower countries, offspring of the Gods Philopatores…

Egyptian

Demotic

Greek

Human

Fly

Bacterium(Plasmid)

Deciphering the language

Percent of Total Number of Types Cell Weight of Each Molecule

Water 70 1 Inorganic ions 1 20 Sugars and precursors 1 250 Amino acids and precursors 0.4 100 Nucleotides and precursors 0.4 100 Fatty acids and precursors 1 50 Other small molecules 0.2 ~300 Macromolecules (proteins, nucleic 26 ~8000 acids, and polysaccharides)

Total ~11000

Bacteria Have ~1e10 Molecules

Non-ideal Cellular EnvironmentA small box 100 nm on a side

~450 proteins~30 ribosomes~340 tRNAsSeveral mRNAs30,000 small organics50,000 ions70% water

In bacteria: 1nM=1 molecule

In E. coli: >80% of proteins < 100 nM

Review of Central Dogma

The Genome of an organism is its

“program”

DNA Codes for RNA

Aacggtggtcgatatgctgagactagctagactacgactttacgactttagcact.

transcript (RNA)

transcription factor

RNAPgene

promoter

RNA codes for Protein

Aacggtggtcgatatgctgagactagctagactacgactttacgactttagcact.

Ribosome Binding Site GeneProtein

Proteins Form Chemical Reaction Networks

Folded Protein

Other Folded Proteins

Organics and ions

Aacggtggtcgatatgctgagactagctagactacgactttacgactttagcact.

DNA and RNA

Synthetic Biology Abstraction

Parts

Devices

Systems Chasses Devices made of devices Applications

Parts

“Atoms” of function

Most commonly: All parts encoded in DNA Some parts are DNA: promoters, etc. Some parts are expressed

RNA Protein

Some parts are synthesized metabolitrd

Promoters

Ribosome Binding Sites

Open Reading Frames

Terminators

Basic Parts

DevicesA GFP Producing Device

tetR RBS GFP Ter. Ter.

Standardization of Part manufacture and the Biobrick Language

Drew Endy Tom Knight

The Parts Registry

http://parts.mit.eduDrew Endy, Tom Knight, Randy Rettberg

Part functions Binding

DNA, Scaffolds, etc. Targeting

Secretion signals (parts on parts) Activity

RNAP activation/inhibition Macromolecular modification:phosphorylation, etc. Metabolite consumption/production Immune response triggering Luminescence/fluorescence Photoenergy conversion Redox …

No common currency really. Classes of mechanisms and physics of function

How many functions/part?

More than we usually think Energetic and time cost of

production is a “function” Non-specific binding is a “function” Uncontroled cross-reaction is a

“function” Promiscuity of activity is a (possibly

very useful) “function.”

Sensors Processors Actuators

Reporter Genes

Signal Integrators

Biosynthetic Genes

Digital SwitchesVirulence Genes

Categorization of parts?

Devices?

Groups of parts that create a new function.

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A possible pulse generator. C-terminal domain of Spo0AComplexed to DNA

Devices?

Groups of parts that create a new function.

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A possible pulse generator.

100 200 300 400

500

1000

1500

2000

Basic function of PG

Time (min)

G fluorescence/OD

Devices?

Groups of parts that create a new function.

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A possible pulse generator.Models of function

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Systems Assemblages of Devices

Fuzzy hierarchy… Systems are large scale devices in

some ways… But also think of them as

applications. Or maybe designs in their

environment.

Device: quorum sensor

Part: anaerobic-activated promoter

Part: adhesion-activated promoter

Part: radiation-activated promoter

Part: amber suppressor

Part: transcriptional activator

Part: two-component sensing domains

Part: adhesion-specificity domains

Part: ribosome binding site

Part: TTSS secretion domain

Device: promoter-driven AND gate

Device: directed motility (Tar chimeras)

Part: invasin

Device: intracellular timer

Device: Effector-less TTSS needle for heterologous protein secretion into eukaryotic cytosol

Chassis: modified lipid A to reduce septic shock

Chassis: E. coli chassis with reduced genome

Testbed: tumor-targeting microbe

Systems Design

Systems Design

Define Problem “Modularize Solution” Selection things to sense, logic, and actuators

needed Select/Design chassis Select/Design parts Predict/Measure how composition works Make many test compositions at once. (library) Measure performance of pieces and systems

at different levels (how?) Try again (and again, and again, and…)

Classical metabolic engineeringGoal: produce more arginine

Ubiquitous pathway(often we are making new chemicals)

How do we make more?

Strategies Overexpress New pathways for

production of L-glutamate and acetyl-CoA

More active enzymes from other species.

Removal of bleed-off of other pathways

The context is important.

Regulation and Timing is Critical

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Systems Questions 1

Is arginine biosynthesis “factorable” Can it be buffered from the cell’s own

metabolism (not likely) How critical is it’s regulation? Will it be maintained in a bioreactor?

(selective pressure) Is it cost effective, scalable, uncontaminable?

This is just for the classical work… What if our systems are WAY more

complicated?

Systems 2: Engineering Beyond the Bioreactor

Therapeutic Bacteria and Viruses Remediation of water, soil and air

by engineered microbes Soil management and agricultural

optimization with microbial and plant GMOs

Environmental sensing and “Plant” protection (naval ship hulls, oil pipelines, etc.)

Systems 2: Engineering Beyond the Bioreactor

Engineering for complex behavior

Uncertainty in the operation of the system and in the environment

Engineering for ecological stability and minimal perturbation

Engineering for safety and containment

Cause of “Hamburger Disease”Enterohaemorrhagic E. coli (EHEC)

“Devices” of Bacterial Pathogenesis

LEE Cluster: Formation of Adhering/Effacing Lesions upon adhesion

bfpA Locus: Adhesion via bundle-forming pili

Stx1 prophage: Secrete shiga toxin to induce haemorrhaging

Complex Regulation of Bacterial Virulence

Genetic Regulation: Controls the when, where, and how much of gene expression

Sensing in the LEE Cluster

pH gastrointestinal location

autoinducer bacterial density

membrane control surface proteinstress expression

Epinephrine host state

ppGpp starvation

Control of Gene Timing

Cascades of Gene Expression

A Snapshot of Regulation at a Complex Promoter

Multiple regulatory proteins can interact with each other, small molecules, and the DNA to induces changes in transcription or translation

Other regulatory elements affect protein activity post-translationally

DNA Binding

A protein-protein interaction

DNA Upstream of LEE Genes

DNA Binding

Challenges

Complexity is higher Factorization not as obvious Uncertainty in mechanisms much

higher Measurements and selections are

much harder Risks higher Selection and “sex” more of a

problem.

Needs

Multiscale Design High throughput construction of parts and

systems. Design for abstraction and predictability Experimental Testing Risk Assessment framework.

Agenda

Classical Manufacture Parts: Protein design Devices: Scaffold design Systems: Therapeutic Bacteria Characterization Future Manufacture

Wrap up.