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
Jun 09, 2015
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