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Centre of Exellence. DNA guided assembly line. Rok Gaber Laboratory of Biotechnology. The 2nd International Symposium Vera Johanides Biotechnology in Croatia by 2020 11.5.2013. Many useful compounds are produced by natural microorganisms. …. Biomaterials . - PowerPoint PPT Presentation
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DNA guided assembly lineRok Gaber
Laboratory of Biotechnology
Centre of Exellence
The 2nd International Symposium Vera JohanidesBiotechnology in Croatia by 2020
11.5.2013
Many useful compounds are produced by natural microorganisms
…Biomaterials. Biologically
active substances.
Pigments,Antioxidants,
Food supplements.
More.Biofuels.
2
3
Enzyme
1
Enzyme
2
Enzyme
3
Enzyme
4
Inovative approach - scaffolding of biosynthetic pathway enzymes
4
Enzyme
1
Enzyme
2
Enzyme
3
Enzyme
4
Inovative approach - scaffolding of biosynthetic pathway enzymes
5
Inovative approach - scaffolding of biosynthetic pathway enzymes
• Direct covalent linkeage of succesive enzymes
• Introduction of protein interacting domains
• Protein scaffolding• RNA scaffolding• DNA scaffolding
6
Selection of DNA binding domains
• Sequence specific• High affinity• Similar characteristics• Available in large number
Zinc finger Leucine zipper Helix-turn-helix
7
Testing of selected Zinc Fingers
Zinc Finger
pBAD
-35 -10ZFBS Reporter gene
RNA polymerase
-35 -10ZFBS Reporter gene
RNA polymerase
ZF
X X
XZinc finger expression is not induced.
Zinc Finger
pBAD
Zinc finger expression is induced.
8
Testing of Zinc Fingers
9
Proof of principle
• Can Zinc Fingers simultaneously bind to adjacent binding sites?
10
Proof of principle
• Assay 1:– DNA enabled split fluorescent protein reconstitution.
2bp
N-YFP C-YFP
11
Proof of principle
• Assay 1:– DNA enabled split fluorescent protein reconstitution.
12
Model biosynthetic pathway – resveratrol biosynthesis
• Enzymes from two different organisms• Direct covalent linkeage of 4CL and STS increases
production of resveratrol (Zhang et al. 2006).• Extendable biosynthetic pathway
TAL OMT
13
Design of Zinc Finger – enzyme fusions
TAL OMT
2bp 4bp 8bp16X ( ) ( () )( )
14
Final results
4CL::S
TS fusio
n
no program
2bp sp
acer
4bp sp
acer
8bp sp
acer
0
2
4
6
8B
resv
erat
rol (
mg/
l)
2h 6h 27h
0
5
10
15
4CL::STS fusion
no program
2bp spacer
4bp spacer
8bp spacer
C
resv
erat
rol (
mg/
l)
3-fold increase of resveratrol production in presence of DNA scaffold.
Minutes0 5 10 15 20 25 30 35
mA
U
0
10
20
30
40
50
Minutes0 5 10 15 20 25 30 35
mA
U
0
10
20
30
40
50
Minutes0 5 10 15 20 25 30 35
mA
U
0
10
20
30
40
50
Standards
No program
2bp spacer
p-co
umar
ic a
cid
tran
s-re
sver
atro
l
pter
ostil
bene
15
Acknowledgements and contributions
iGEM 2010 team Slovenia
Jernej Turnšek Nejc TomšičTjaša Stošicki Tina IlcTina Lebar Matej
ŽnidaričMattia Petroni Jure
BordonRok Pustoslemšek Rok Črešnovar
Miha Mraz Miha Moškon
Nikolaj Zimic
Monika AvbeljJerneja Mori Jernej Turnšek
Mojca BenčinaGregor Anderluh Irena VovkMateja Puklavec
Tomaž KoprivnjakRok Gaber Tina Lebar
Vesna Hodnik Vesna Glavnik Breda Simonovska
Roman Jerala