Synthetic Biology

announcing the advancing

technological revolution

Roman Jerala

Department of biotechnology

National institute of chemistry, FKKT UL

EN-FIST Centre of Excellence

Ljubljana, Slovenia

The miracle of a Seed

DNA as the blueprint of life

2001 Draft of the

human genome

Genome sequencing

1000 genomes project initiated in 2008 60x more sequence data than combined previous 25 years

Information stored in the DNA

How much of DNA is on the world:

• 4*1035 bp bacterial DNA (5% DNA)

• 4*1034 bp plant DNA (1% DNA)

• 1*1034 bp animal DNA (1% DNA)

• 5*1030 bp viral DNA (50% DA)

• 5*1035 bp of DNA on Earth

~1 bit/nm3, much higher density than the existing information storage media

Genome could be rewritten or modified

Chemical synthesis of

oligonucleotides

annealing

ligation

Insertion into plasmid

Introduction

into the cell

The assembly of a synthetic M. mycoides

genome

D G Gibson et al. Science 2010;329:52-56

Assembled in yeast from 1078

overlapping DNA cassettes in three

steps:

1. 1080 bp casettes from synthetic

oligos for 10 kbp assemblies

2. 10 assemblies were combined into

100 kbp assemblies

3. 11 fragments were combined into

the full chromosome

Metabolic pathways of a cell

The engineering approach of

synthetic biology

• Application of engineering principles into biological systems

• Important engineering principles: – Modularity

– Abstraction

– Reliability

– Predictability

– Standardization

Adriananoandro et el., Mol.Sys.Biol. 2006

(Synthetic) Biology is

nanotechnology that works. Drew Endy

http://www.arn.org/mm/mm.htm

Synthetic biology as an

investigative tool

How and why are natural systems

constructed as they are ?

(functional requirements or

evolutionary coincidence)

Do we understand the function of all

components of a device/system?

“What I can not create, I do not

understand!”

Richard Feynman

Synthetic biology contributes

to solve problems in

• Health/Medicine

• Sustainable sources of energy

• New materials & bionanomaterials

• Information processing

• Biosensors

• Bioremediation...

Področja uporabe sintezne biologije

Synthetic biology to

(re)design products

More efficient and

sustainable,

production:

-Drugs to treat malaria

-Biofuels

-...

http://keaslinglab.lbl.gov/application_areas/index.html

Complex biosynthetic pathways

Enzyme

1

Enzyme

2

Enzyme

3

Enzyme

4

Limitation of multienzyme biosynthetic pathways

Enzyme

1

Enzyme

2

Enzyme

3

Enzyme

4

Scaffolding of biosynthetic pathway enzymes to enhance the biosynthetic rate

fusion proteins between DNA

binding domain and functional

domain (e.g. enzymes)

Bound enzymes perform sequential reactions

the order of DNA motifs along the

program DNA defines the order of

bound functional proteins

Improved biosynthesis using DNA scaffold

Improved biosynthesis using DNA scaffold

4CL STS

2h 6h 27h

0

5

10

15

4CL::STS fusion

no program

2bp spacer

4bp spacer

8bp spacer

C

resvera

tro

l (m

g/l)

300% yield improvement

Renewable sources of energy

CO2

cellulose

sugars

fuel

Microbial

conversion

enzymes

Sintezna biologija za obnovljive vire energije

CO2

Logic-gated nanorobot for drug

delivery

Douglas et al., Science 2012

DNA origami forms a

hexagonal barrell split in

half

Complementary clamps

close the barrell

Binding sites inside the

barrel can be loaded with

cargo conjugated to

ssDNA

Binding to cells depending on the

combination of surface-exposed markers

Nanostructures in nature

Transcending the nature

Can we bend molecules at will ?

Polypeptide origami

• Polypeptides fold into defined tertiary structures

• Nature solved the design problem during hundreds of millions

of years of evolution

Design of complex 3D structures

Deconstruct the designed

structure into smaller rigid

independently-folded building

elements

and

Concatenate those elements

into the self-assembling

polypeptide

http://bestbridge.net/Eu_en/the-iron-bridge.html

Coiled-coil

interactions governing the specificity of coiled-coil formation

are well understood

coiled-coils are characterized by regular repeating unit of 7 amino acids heptad repeat (a-b-c-d-e-f-g)

stability and specificity is provided by residues at defined positions within the heptad repeat

How can we build complex structures from rigid building blocks ?

Flexible linker between building elements

Each edge in the final structure is composed of a dimer

Flexible linker between building elements

Design of a tetrahedron-forming polypeptide

TET12

4 parallel dimers

2 antiparallel dimers APHP3BCRGCNshAPHP7GCNshP4P5P8BCRP6

flexible tetrapeptide linker

His6

Polypeptide production, isolation, assembly

SDS-PAGE

Production in E.coli Self-assembly

Dialysis at low polypeptide

concentration

Protein purification

Affinity chromatography HPLC-RP

Detection of nano-tetrahedra

Negative staining

Nanogold 2 nm + Negative staining

HHHHHH

N- an C-terminal ends of the tetrahedral path coincide

“See things not as they are, but as they

might be”

Robert Oppenheimer

Watermark in the synthetic genome of Mycoplasma laboratorium

Slovenian teams 2006-2010

Mentors Mojca Benčina, Monika Avbelj, Karolina Ivičak , Nina Pirher, Gabriela Panter, Mateja

Manček Keber (KI), Marko Dolinar(FKKT), Simon Horvat (BF), Iva Hafner Bratkovič, Helena Gradišar, Ota Fekonja, Jelka Pohar, Rok Gaber, Tomaž Koprivnjak, Jerneja Mori, Irena Vovk, (KI), Gregor Anderluh, Vesna Hodnik (BF) , Miha Mraz, Miha Moškon, Nikolaj Zimic (FRI), Roman Jerala (KI, FKKT)

2008

Eva Čeh, BF

Vid Kočar, FKKT

Katja Kolar, FKKT

Ana Lasič, MF

Jan Lonzarić, FKKT

Jerneja Mori, BF

Anže Smole, BF

2006

Monika Ciglič, BF

Ota Fekonja, BF

Jernej Kovač, FKKT

Alja Oblak, BF

Jelka Pohar, BF

Matej Skočaj, BF

Rok Tkavc, BF

2007

Marko Bitenc, BF

Peter Cimermančič, FKKT

Rok Gaber, BF

Saša Jereb , FKKT

Katja Kolar, FKKT

Anja Korenčič, FKKT

Andrej Ondračka, FKKT

2009

Sabina Božič, FKKT

Nika Debeljak, BF

Tibor Doles, BF

Urška Jelerčič;FMF

Anja Lukan, FKKT

Špela Miklavič, BF

Marko Verce, BF

2010

Jernej Turnšek

Nejc

Tjaša

Tina Ilc

Tina Lebar

Matej Žnidarič

Mattia Petroni

Jure Bordon

Rok Pustoslemšek

Rok Črešnovar