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