Roman Jerala, National Institute of Chemistry, Ljubljana, Slovenia Synthetic biology announcing the coming technological revolution http://obc2012.outofthebox.si/

1. 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

3. The miracle of a Seed

4. DNA as the blueprint of life

5. Genome sequencing
2001 Draft of the
human genome
1000 genomes project initiated in 2008
60x more sequence data than combined previous 25 years

6. 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

7. Chemical synthesis of
oligonucleotides
annealing
ligation
Insertion into plasmid
Introduction
into the cell

8. The assembly of a synthetic M. mycoides
genome
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
D G Gibson et al. Science 2010;329:52-56

9. Metabolic pathways of a cell

10. 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

11. (Synthetic) Biology is
nanotechnology that works.
Drew Endy
http://www.arn.org/mm/mm.htm

12. Synthetic biology as an
investigative tool
“What I can not create, I do not
understand!”
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?
Richard Feynman

13. Področja uporabe sintezne biologije
Synthetic biology contributes
to solve problems in
• Health/Medicine
• Sustainable sources of energy
• New materials & bionanomaterials
• Information processing
• Biosensors
• Bioremediation...

14. Synthetic biology to
(re)design products
More efficient and
sustainable,
production:
-Drugs to treat malaria
-Biofuels
-...
http://keaslinglab.lbl.gov/application_areas/index.html

15. Complex biosynthetic pathways

16. Limitation of multienzyme biosynthetic pathways
Enzyme Enzyme
3 4
Enzyme Enzyme
2 1

17. Scaffolding of biosynthetic pathway enzymes to
enhance the biosynthetic rate
Enzyme Enzyme Enzyme Enzyme
3 4 1 2

18. Improved biosynthesis using DNA scaffold
the order of DNA motifs along the
program DNA defines the order of
bound functional proteins
fusion proteins between DNA
binding domain and functional
domain (e.g. enzymes)
Bound enzymes perform sequential reactions

19. Improved biosynthesis using DNA scaffold
300% yield improvement
15
C
resveratrol (mg/l)
10
4CL STS
5
0
2h
6h
h
27
4CL::STS fusion 4bp spacer
no program 8bp spacer
2bp spacer

20. Sintezna biologija za obnovljive vire energije
Renewable sources of energy
CO2
enzymes
cellulose
sugars
CO2
Microbial
conversion
fuel

21. Logic-gated nanorobot for drug
delivery
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
Douglas et al., Science 2012

22. Binding to cells depending on the
combination of surface-exposed markers

23. Nanostructures in nature
Transcending the nature
Can we bend molecules at will ?

24. Polypeptide origami
• Polypeptides fold into defined tertiary structures
• Nature solved the design problem during hundreds of millions
of years of evolution

25. 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

26. 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

27. Flexible linker between building elements
 How can we build complex structures from rigid building blocks ?

28. Flexible linker between building elements
Each edge in the final structure is composed of a dimer

30. Design of a tetrahedron-forming polypeptide
4 parallel dimers
APHP3BCRGCNshAPHP7GCNshP4P5P8BCRP6
2 antiparallel dimers
TET12 His6
 flexible tetrapeptide linker

31. Polypeptide production, isolation, assembly
Production in E.coli Self-assembly
SDS-PAGE
 Dialysis at low polypeptide
concentration
Protein purification
Affinity chromatography
HPLC-RP

32. Detection of nano-tetrahedra
HHHHHH
Negative staining
Nanogold 2 nm
+ Negative staining

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

34. “See things not as they are, but as they
might be”
Robert Oppenheimer
Watermark in the synthetic genome of Mycoplasma laboratorium

35. Slovenian teams 2006-2010
2007 2008 2009 2010
2006
Marko Bitenc, BF Eva Čeh, BF Sabina Božič, FKKT Jernej Turnšek
Monika Ciglič, BF
Peter Cimermančič, FKKT Vid Kočar, FKKT Nika Debeljak, BF Nejc
Ota Fekonja, BF
Rok Gaber, BF Katja Kolar, FKKT Tibor Doles, BF Tjaša
Jernej Kovač, FKKT
Saša Jereb , FKKT Ana Lasič, MF Urška Jelerčič;FMF Tina Ilc
Alja Oblak, BF
Katja Kolar, FKKT Jan Lonzarić, FKKTAnja Lukan, FKKT Tina Lebar
Jelka Pohar, BF
Anja Korenčič, FKKT Jerneja Mori, BF Špela Miklavič, BF Matej Žnidarič
Matej Skočaj, BF
Andrej Ondračka, FKKT Anže Smole, BF Marko Verce, BF Mattia Petroni
Rok Tkavc, BF
Jure Bordon
Rok Pustoslemšek
Rok Črešnovar
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)