iGEM Slovenia @ 6th CeBiTec conference

Slovenian Projects
Jernej Turnšek
Department of Biotechnology

Follow me on Twitter: @SynEnthu

6th CeBiTec Symposium Bielefeld, July 20th, 2011

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

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


[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


[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


[regulated nanomaterial assembly]
GyrB

assembly with
coumermycin

disassembly with
novobiocin
CutA1

Potential applications for
drug delivery and
controlled release. 200 nm
TEM image


[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.


[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

DNA coding beyond triplets

iGEM 2010 Team Slovenia

Rethinking the central biological dogma


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.


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


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.

ZNF binding to target DNA in vivo

ZNFs act as artificial repressors.


Assembly of multiple DNA binding domains


Resveratrol biosynthesis
 2-step biosynthetic pathway

4CL: 4-coumarate:CoA ligase

STS: stilbene synthase

 antioxidant
 French paradox


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

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


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...


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


Human practice – make it understandable


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)


iGEM Slovenia @ 6th CeBiTec conference

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