Jernej Turnšek presented on Slovenian iGEM projects from 2006 to 2010 and a 2010 project on using DNA as a programmable scaffold. The key points are: 1) Slovenian iGEM teams won gold medals 5 times between 2006-2010 and were finalists or received best in area awards on other occasions. 2) The 2010 project explored using zinc finger proteins fused to enzymes and bound to DNA to guide multi-step biosynthesis, achieving higher resveratrol yields than a direct fusion. 3) The DNA programmable scaffold approach offers potential for complex biosynthesis pathways and other applications by organizing multiple enzymatic reactions in a defined order.

1. Slovenian Projects
Jernej Turnšek
Department of Biotechnology
Follow me on Twitter: @SynEnthu
6th CeBiTec Symposium Bielefeld, July 20th, 2011

2. Agenda
1) Slovenian iGEM timeline
2) nanoBRICKS[pro] – 2009 project
3) DNA coding beyond triplets – 2010 project
4) Slovenian iGEM teams 2006 – 2010
5) Track record
6th CeBiTec Symposium Bielefeld, July 20th, 2011

3. Slovenian iGEM timeline
2006 – Engineered Human Cells – SAY NO TO SEPSIS
2007 – virotrap® – A Synthetic Biology Approach Against HIV
2008 – Immunobricks
2009  nanoBRICKS[pro]
2010  DNA coding beyond triplets
6th CeBiTec Symposium Bielefeld, July 20th, 2011

5. [aim]
 create self-assembling polypeptide nanostructures
Regulated nanomaterial assembly
Only 0.22um PVDF 0.22um PVDF Filter
Filter with protein membrane
PVDF filter PVDF filter Protein
Polypeptide membranes
with 0.22 with 0.22 membrane
um pores um pores
Coiled-coil-based nanostructure assembly
6th CeBiTec Symposium Bielefeld, July 20th, 2011

6. [strategies for polypeptide self-assembly]
BUILDING BLOCKS
OLIGOMERISATION DOMAINS COILED-COIL SEGMENTS
(dimer, trimer, tetramer, pentamer, hexamer...) (as rigid rods)
 2-7 intertwined α-helices
 8% of natural proteins
 helices in (anti)parallel
orientation
functional self-assembling element
6th CeBiTec Symposium Bielefeld, July 20th, 2011

7. [regulated nanomaterial assembly]
GyrB
assembly with
coumermycin
disassembly with
novobiocin
CutA1
Potential applications for
drug delivery and
controlled release. 200 nm
TEM image
6th CeBiTec Symposium Bielefeld, July 20th, 2011

8. [polypeptide membranes]
p53
APH, APH1 or BCR
- size defined mainly by the length of the coiled-coil segment
- residues at the surface of CC segment define its chemical properties
Potential applications for filtration and separation devices, catalysis.
6th CeBiTec Symposium Bielefeld, July 20th, 2011

9. [coiled-coil-based nanostructure assembly]
- 2 coiled-coils  only 1D fibrils
- 3 coiled-coils  2D and 3D nano-objects
Parallel homodimeric and two heterodimeric segments
a b a’
polypeptide nanocube
polypeptide network
6th CeBiTec Symposium Bielefeld, July 20th, 2011

10. DNA coding beyond triplets
iGEM 2010 Team Slovenia

11. Rethinking the central biological dogma
6th CeBiTec Symposium Bielefeld, July 20th, 2011

12. DNA guides the assembly of functional proteins
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)
Functional proteins perform sequential operations.
6th CeBiTec Symposium Bielefeld, July 20th, 2011

13. Selection of DNA binding domains
 helix-turn-helix, leucine zipper, zinc finger
 modular design
 >700 characterised ZNFs already available in ZiFDB
 262.144 possible binding sites for nonameres (49)
 longer recognition sequence  more specific binding
6th CeBiTec Symposium Bielefeld, July 20th, 2011

14. Characterisation of ZNFs
Gli1, HIVC, Zif268, PBSII, Jazz, Blues
 cloned  expressed  purified  characterized
Surface plasmon resonance (SPR) EMSA
binding
dissociation
Resp. Diff. [RU]
time [s]
Time [s]
All isolated ZNFs bind to their target DNA.
6th CeBiTec Symposium Bielefeld, July 20th, 2011

15. ZNF binding to target DNA in vivo
ZNFs act as artificial repressors.
6th CeBiTec Symposium Bielefeld, July 20th, 2011

16. Assembly of multiple DNA binding domains
6th CeBiTec Symposium Bielefeld, July 20th, 2011

17. Resveratrol biosynthesis
 2-step biosynthetic pathway
4CL: 4-coumarate:CoA ligase
STS: stilbene synthase
 antioxidant
 French paradox
6th CeBiTec Symposium Bielefeld, July 20th, 2011

18. DNA-guided resveratrol biosynthesis
a: Zif268 + E1: 4CL = Zif268::4CL fusion
b: PBSII + E2: STS = PBSII::STS fusion
D 50
40
4CL::STS
30 *
mAU
20
 length of the DNA program: 16x scaffold with 2, 4 or 8bp spacer 10
 compared to direct fusion 4CL::STS and no DNA program 0
0 5 10 15 20 25 30 35
Minutes
D 50 50
40
4CL::STS 40
2bp spacer
30 * 30
mAU
mAU
20 20
*
10 10
0 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Minutes Minutes
50
6th CeBiTec Symposium Bielefeld, July 20th, 2011

19. Results summary
8 15
B C
resveratrol (mg/l)
resveratrol (mg/l)
6
10
4
5
2
0
0
2h
6h
h
27
m
r
r
er
on
4b ace
8b ace
ra
ac
4CL::STS fusion 4bp spacer
si
og
sp
sp
sp
fu
pr
TS
p
p
p
no program 8bp spacer
2b
no
:S
L:
2bp spacer
4C
6th CeBiTec Symposium Bielefeld, July 20th, 2011

20. Conclusions
• we invented a new type of ordered protein
assembly based on a DNA program
8
• exciting potentials for biosynthesis
B
resveratrol (mg/l)
6
• yield improvement 4
• chanelling 2
0
m
r
er
er
on
4b ace
ra
ac
ac
si
og
sp
sp
sp
fu
pr
TS
p
p
p
2b
8b
no
:S
L:
• 151 BioBricks submitted to the Registry:
4C
universal DNA binding domains testing
device, split GFPs, zinc fingers...
6th CeBiTec Symposium Bielefeld, July 20th, 2011

21. Future perspectives
• complex (and multiple) biosynthetic
pathways with several reactions (e.g. drugs,
biofuels…)
• in vitro scaffold for multienzyme reactions
Beyond biosynthesis:
• pattern recognition
• information processing (using protein kinases,
proteases etc.)
Keasling et al., 2003, Nature Biotechnology
6th CeBiTec Symposium Bielefeld, July 20th, 2011

22. Human practice – make it understandable
6th CeBiTec Symposium Bielefeld, July 20th, 2011

23. Slovenian iGEM teams 2006-2010
2006 2007 2008 2009 2010
Monika Ciglič Marko Bitenc Eva Čeh Sabina Božič Jernej Turnšek
Ota Fekonja Peter Cimermančič Vid Kočar Nika Debeljak Nejc Tomšič
Jernej Kovač Rok Gaber Katja Kolar Tibor Doles Tjaša Stošicki
Alja Oblak Saša Jereb Ana Lasič Urška Jelerčič Tina Ilc
Jelka Pohar Katja Kolar Jan Lonzarić Anja Lukan Tina Lebar
Matej Skočaj Anja Korenčič Jerneja Mori Špela Miklavič Matej Žnidarič
Rok Tkavc Andrej Ondračka Anže Smole Marko Verce Mattia Petroni
Jure Bordon
Rok Pustoslemšek
Rok Črešnovar
Mentors
Mojca Benčina, Monika Avbelj, Karolina Ivičak, Nina Pirher, Gabriela Panter, Mateja Manček Keber, Iva Hafner Bratkovič, Helena
Gradišar, Ota Fekonja, Jelka Pohar, Rok Gaber, Tomaž Koprivnjak, Jerneja Mori, Irena Vovk (KI), Marko Dolinar (FKKT), Simon Horvat
(BF, KI), Gregor Anderluh (KI, BF), Vesna Hodnik (BF), Miha Mraz, Miha Moškon, Nikolaj Zimic (FRI), Roman Jerala (KI, FKKT, En-FIST)
6th CeBiTec Symposium Bielefeld, July 20th, 2011

24. Sponsors
6th CeBiTec Symposium Bielefeld, July 20th, 2011

25. Track record 2006 – 2010
 Gold medal: 5x
 Finalists: 4x
 Best in Area: 4x
 Grand Prize: 3x (2006, 2008, 2010)
6th CeBiTec Symposium Bielefeld, July 20th, 2011

26. Questions & Answers

27. [comparison of DNA and polypeptides as nanostructure building blocks]
DNA polypeptides
contains 4 nucleotides with similar contain 20 AA with different chemical
properties [-] properties [+]
can fold into defined 3D structures
can fold into defined 3D structure [+]
[+/-]
build structures and functional devices
used to store information in nature [-]
in nature [+]
structure-encoding information is
easy to program (W-C base pairs) [+]
complex [-]
6th CeBiTec Symposium Bielefeld, July 20th, 2011

28. Protein scaffold vs. DNA scaffold
DNA scaffold
Protein
scaffold
Spatial orientation Bundled Linear
Order Unpredictable Highly predictable order
Scaffold: binding Similar, well characterized
Variations in strength
domain interactions interactions
Limited number of Large number of DNA
Number of available
protein dimerization binding domains is readily
elements
domains available available
6th CeBiTec Symposium Bielefeld, July 20th, 2011

29. Role of the spacer length
6th CeBiTec Symposium Bielefeld, July 20th, 2011

30. Confirmation of FRET
Photobleaching of the acceptor molecule (YFP).
Quantification of the FRET effect upon the addition of a DNA program.
Four zinc fingers linked to funtional domains can bind simultaneously to
neighbouring sites on DNA
6th CeBiTec Symposium Bielefeld, July 20th, 2011