F Freiburg_bioware ib bi Universal endonuclease cutting edge - - PowerPoint PPT Presentation

F ib bi Freiburg_bioware

Universal endonuclease ‐ cutting edge technology g g gy

General Idea

Programmable Restriction Endonuclease g

Motivation: T t i ti

• Too many restriction enzymes
• Short recognition sites
• Too many cuts in genomes

y g

• Many cloning RFC‘s
• No in vivo application

Idea:

• One programmable oligonucleotide

p g g

• One enzyme
• Infinite possibilities

FokI FokI

In Vitro Cloning: PCR‐like procedure g p

5’ 3’

FokI Based Structure Modelling

Heterodimerization of Cleavage Domains

TypIIS restriction endonuclease FokI

• Separation of FokI

cleavage domains cleavage domains

WT WT

• Inactivation through

Fok_i Fok_a Fok_i Fok_a

Inactivation through exchange of catalytical active amino acids

• Heterodimerization
• Heterodimerization

via exchange of amino acids

Linkage to Anticalins & Programmable Adapter

Fok_i Fok_a Fluorescein‐binding anticalin Digoxigenin‐binding anticalin FluA DigA anticalin anticalin Fluorescein Digoxigenin Fluorescein Digoxigenin

Programmable Restriction Endonuclease

target 5' C C C T T A T G A T T G A C C G T C T G C G C C T C G T T C C G G C T A A G T A A C A T G G guide 3' G G G A A T A C T A A C T G G C A G A C G C G G A G C A A G G C C G A T T C A T T G T A C C

Di-oligo 16b Di-oligo A 16b Di-oligo 16b Di-oligo B 30b Mono-oligo 30b

Fluorescein Digoxigenin

Cloning Strategy

Cloning Strategy

Fok fragment Tag Binding protein Linker

His FluA Inactive Split His FluA Inactive Short Split Strep Active DigA Middle L Signal Sequence (D bA) Long (DsbA) Active Fok YFP (Venus)

Cloned according to RFC 25, Fusion Protein (Freiburg) Biobrick assembly standard

Cloning Strategy

lacIq/AraC

XbaI

• Conversion of commercial NEB

/

XbaI NgoMIV

• Conversion of commercial NEB

and Invitrogen vectors to iGEM expression vectors including

pEX/ pBad

• ri

NgoMIV

RBS and iGEM restriction sites

AmpR

AgeI SpeI NotI PstI

• Extension for BioBrick standard

expression parts

AgeI SpeI

• RFC 25 cloning implemented in

registry with Randy

lacIq

XbaI p NotI PstI

registry with Randy

XbaI NgoMIV PstI

pJS#419

• ri

AgeI

CmR

AgeI SpeI NotI PstI

Modeling: bind ‐ ready ‐ steady ‐ cut

Modeling: bind ‐ ready ‐ steady ‐ cut

Fok iFok a Fok_iFok_a

k5on k5off

DNA DNA

Fok_i + Fok_a DNAFok i DNAFok a

k1a_on k1a_off k1i_off k1i_on

• _

_

Ready

k2a_on k2a_off k2i_off k2i_on

Steady Fok_i

Fok_a

k3_on k3_off

y

k4

cut

Ordinary Differential Equations

7 ODEs 7 ODEs 13 Parameters Numerically solved y Hi h t t f Highest amount of active enzyme dependent on the p ratio of Fok_active and Fok_inactive If boths affinities differ, the amount of expression has to be adjusted

Protein Expression & Purification

Purification Scheme

Fok_inactive Fok_active Tagged protein His‐tag Strep‐tag GST‐tag Affinity purification Ni NTA S T i Gl hi Ni‐NTA StrepTactin Glutathione dialysis size exclusion

Purification Scheme

Fok_inactive Fok_active Tagged protein Tagged protein His‐tag Strep‐tag GST‐tag tag

Purification Scheme

Fok_inactive Fok_active Affinity purification Ni NTA S T i Gl hi Ni‐NTA StrepTactin Glutathione tag

Purification Scheme

Fok_inactive Fok_active 2nd tag tag 2 tag dialysis size exclusion

Argonaute Protein from Aquifex aeolicus

Ni‐NTA purification size exclusion chromatography Ni NTA purification size exclusion chromatography 80 kDa 58 kDa

His‐FluA‐Linker‐Fok_inactive

Hi t Ni NTA ifi ti 46 kDa

• His‐tag, Ni‐NTA purification

30 kDa

His‐FluA‐Linker‐Fok_inactive

46 kDa Hi t Ni NTA ifi ti 30 kDa

• His‐tag, Ni‐NTA purification
• Western Blot

Western Blot ‐ anti‐His‐HRP antibody 45 kDa

control

specific antibody

control

tag

membrane

Expression Control: Fok_active‐YFP(Venus)

• Fluorescence microscope YFP filter

Expression Control: Fok_active‐YFP(Venus)

• Fluorescence microscope YFP filter
• SDS gel: cell lysate

175 kDa 80 kDa 58 kDa induced/uninduced 58 kDa 46 kDa

Expression Control: Fok_active‐YFP(Venus)

• Fluorescence microscope YFP filter
• SDS gel: cell lysate

175 kDa 80 kDa 58 kDa induced/uninduced 58 kDa 46 kDa 175 kDa 80 kDa

• Western Blot: anti‐YFP

80 kDa 58 kDa 46 kD 46 kDa

In Vivo Assays

Transformation of Labeled Oligos

E coli

• E. coli

Fluorescence microscope image

Phage DNA Cleavage in E. coli

Fok i Fok_a

M13 DNA

Fok_i

E coli Electroporation Cotransformation

• E. coli

Electroporation Plaque assay

IPTG/X‐Gal plates IPTG/X Gal plates Control Test

Control ‐> plaques In vivo activity ‐> no plaques Conclusion: In vivo assay demonstrated cleavage activity of the final Fok constructs

M13 without

• ligo

M13 with oligo

In Vitro Assays: Proof of Principle

910 bp B 218 bp M13 mp18 7249 b A B C

M13 mp18

7249 bp D 3334 bp 2787 bp

mp18

3334 bp 2787 bp

3 kb

910 nt A B

1 kb 0 5 kb

M13 mp18 7249 nt A

0,5 kb

6339 nt 6339 nt

In Vitro Assays

E li

• E. coli

Fok_a

sonication

Fok_i

37°C id DNA

hybridization

target DNA guide DNA 1

80 nt

3‘ 5‘

30 nt 16 nt / 30 nt

2 Cy3

80 nt

3‘ 5‘

2

In Vitro Cleavage Assays

100 bp 75 bp 50 bp 50 bp 25 bp 25 bp

1 2 1 2

5 ‘

t

5‘

80 nt 30 nt

80 nt 16 nt / 30 nt

3 ‘

3‘ 80 nt

3‘ 5‘

30 nt

Guided Fok fusion construct cleaved the ssDNA as expected!

Argonaute Proteins

Argonaute Proteins: DNA Cleavage?

• Key players in RNA interference
• Loaded with siRNA‐>RISC
• Site & sequence specific mRNA cleavage

5‘ Target 3‘ Guide 3‘ Mid N Guide 5‘ Mid N PIWI

ssDNA Cleavage by Argonaute Protein

M13 x x x x x x

• Prot. K

x x x x x x AGO x x x x x Oligo 1 x x

AGO + guide oligo

Oligo 1 x x Oligo 2 x x

AGO guide oligo

30’, 55°C

M13 ssDNA Proteinase K addition

30’, 55°C

Proteinase K addition

Protein Presentation on Phages

Phagemid Fusion protein

FLAG‐tag ELISA

HRP 3,0 4,0

sorption BSA Anti‐FLAG

Anti‐M13 1,0 2,0

05 nm Abs

Anti‐FLAG FLAG‐tag 0,0

no FLAG‐tag FLAG‐tag AGO‐FLAG‐tagAGO‐FLAG‐tag

4

Optimization: Selection System

Oligo

Streptavidin Biotinylated oligo

1 2

Phage presenting AGO

1:Incubation with oligo Ca2+ 3 :Incubation with oligo 2:Binding to target 3:Washing of non binding 4:Ca2+ enables catalysis Ca 4 4:Ca2+ enables catalysis 5:DNaseI elutes rest After two rounds of panning several promising clones were isolated

DNaseI

5 promising clones were isolated

Summary

• Conceived idea of a universal restriction enzyme.

Conceived idea of a universal restriction enzyme.

• Modeled 3D structure and the reaction kinetics.

d l l l d di C2 ( ib f i d d)

• Modularly cloned according to RFC25 (Freiburg fusion standard)
• Expressed and purified proteins.
• Guided in vitro cleavage was demonstrated.
• In vivo activity was shown by a phage assay.

In vivo activity was shown by a phage assay.

• An international ethics survey was conducted. >300 people in 13 countries

i i d were interviewed

• >35 parts were submitted and well documented. Scar issue for RFC25 was

resolved within the BioBrick registry. Previous parts were updated.

Acknowledgements

Instructors Support & Instrumentation Instructors Support & Instrumentation

• Dr. Kristian Müller (Biology, Bioss)
• Dr. Stefan Juranek (Rockefeller Institute, New York)
• Dr. Katja Arndt (Biology, FRIAS, Bioss)
• Prof. Dr. Ralf Baumeister (Biology, ZBSA, FRIAS)

Tobias Baumann (Bioss)

• Prof. Dr. Ralf Reski (Biology)

( ) ( gy) Sven Hagen (FRIAS) Freiburg iGEM Team 2008 Janina Speck (Biology) Collaboration iGEM Team Freiburg_software

Ethics: International Survey

Ethics: Results

• Huge lack of understanding
• Negative, skeptical outlook
• Genetically‐modified food not

Genetically‐modified food not appreciated

• Confidence concerning medical

research