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

F Freiburg_bioware ib bi Universal endonuclease ‐ cutting edge technology g g gy

General Idea

Programmable Restriction Endonuclease g Motivation: • T Too many restriction enzymes t i ti • 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 Fok_a Fok_i Fok_i Fok_a • Inactivation through Inactivation through exchange of catalytical active amino acids • • Heterodimerization Heterodimerization via exchange of amino acids

Linkage to Anticalins & Programmable Adapter Fluorescein ‐ binding Fok_i Fok_a Digoxigenin ‐ binding anticalin anticalin anticalin anticalin DigA FluA Fluorescein Fluorescein Digoxigenin 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 Tag Binding protein Linker Fok fragment His His FluA FluA Split Split Inactive Inactive Short Strep DigA Middle Active Signal Sequence L Long (D bA) (DsbA) Active Fok YFP (Venus) Cloned according to RFC 25, Fusion Protein ( Freiburg ) Biobrick assembly standard

Cloning Strategy XbaI lacI q /AraC / • • Conversion of commercial NEB Conversion of commercial NEB XbaI and Invitrogen vectors to iGEM NgoMIV expression vectors including RBS and iGEM restriction sites pEX/ NgoMIV ori pBad • Extension for BioBrick standard AgeI SpeI expression parts NotI AgeI Amp R PstI SpeI p NotI PstI • RFC 25 cloning implemented in lacI q registry with Randy registry with Randy XbaI XbaI NgoMIV ori pJS#419 PstI AgeI AgeI SpeI NotI Cm R PstI

Modeling: bind ‐ ready ‐ steady ‐ cut

Modeling: bind ‐ ready ‐ steady ‐ cut Fok iFok a Fok_iFok_a k5off k5on DNA Fok_i + Fok_a DNA k1a_on k1a_off k1i_on k1i_off DNA Fok a DNAFok i o _ _ k2a_on k2i_on Ready k2a_off k2i_off Fok_i Fok_a k3_on k3_off Steady y k4 cut

Ordinary Differential Equations 7 ODEs 7 ODEs 13 Parameters Numerically solved y Highest amount of Hi h t t f 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 Ni NTA StrepTactin S T i Gl Glutathione hi size dialysis exclusion

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

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

Purification Scheme Fok_inactive Fok_active 2 nd tag 2 tag tag size dialysis exclusion

Argonaute Protein from Aquifex aeolicus Ni ‐ NTA purification Ni NTA purification size exclusion chromatography size exclusion chromatography 80 kDa 58 kDa

His ‐ FluA ‐ Linker ‐ Fok_inactive 46 kDa • Hi t His ‐ tag, Ni ‐ NTA purification Ni NTA ifi ti 30 kDa

His ‐ FluA ‐ Linker ‐ Fok_inactive 46 kDa • Hi t His ‐ tag, Ni ‐ NTA purification Ni NTA ifi ti 30 kDa • Western Blot Western Blot ‐ anti ‐ His ‐ HRP antibody specific antibody 45 kDa control control tag membrane

Expression Control: Fok_active ‐ YFP(Venus) • Fluorescence microscope YFP filter

Expression Control: Fok_active ‐ YFP(Venus) • Fluorescence microscope YFP filter 175 kDa 80 kDa • SDS gel: cell lysate 58 kDa 58 kDa induced/uninduced 46 kDa

Expression Control: Fok_active ‐ YFP(Venus) • Fluorescence microscope YFP filter 175 kDa 80 kDa • SDS gel: cell lysate 58 kDa 58 kDa induced/uninduced 46 kDa 175 kDa 80 kDa 80 kDa 58 kDa • Western Blot: anti ‐ YFP 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_a Fok i Fok_i M13 DNA Cotransformation Electroporation Electroporation E coli E. coli Plaque assay IPTG/X Gal plates IPTG/X ‐ Gal plates Control In vivo activity Control Test ‐ > plaques ‐ > no plaques Conclusion: In vivo assay demonstrated cleavage activity of the final Fok constructs M13 without M13 with oligo oligo

In Vitro Assays: Proof of Principle 218 bp 910 bp B B C A M13 mp18 M13 mp18 mp18 7249 b 7249 bp D 2787 bp 2787 bp 3334 bp 3334 bp 3 kb 910 nt B A A 1 kb M13 mp18 7249 nt 0 5 kb 0,5 kb 6339 nt 6339 nt

In Vitro Assays E E. coli li Fok_a Fok_i sonication 37°C hybridization guide DNA id DNA 30 nt 1 target DNA 3‘ 5‘ 80 nt 16 nt / 30 nt 2 2 3‘ 5‘ 80 nt Cy3

In Vitro Cleavage Assays 100 bp 75 bp 50 bp 50 bp 25 bp 25 bp 1 2 1 2 ‘ 5‘ 5 30 nt t 16 nt / 30 nt 80 nt 80 nt 30 nt ‘ 3 3‘ 5‘ 3‘ 80 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‘ 3‘ Target 3‘ Guide Guide 5‘ N N Mid Mid 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 AGO guide oligo AGO + guide oligo Oligo 1 Oligo 1 x x x x Oligo 2 x x 30’, 55°C M13 ssDNA 30’, 55°C Proteinase K addition Proteinase K addition

Protein Presentation on Phages Phagemid Fusion protein FLAG ‐ tag ELISA 4,0 sorption BSA 3,0 HRP Anti ‐ FLAG 05 nm Abs 2,0 Anti ‐ M13 1,0 4 0,0 FLAG ‐ tag no FLAG ‐ tag FLAG ‐ tag AGO ‐ FLAG ‐ tagAGO ‐ FLAG ‐ tag Anti ‐ FLAG

Optimization: Selection System Streptavidin Oligo Biotinylated oligo 1 2 Phage presenting AGO 1 :Incubation with oligo :Incubation with oligo 3 2 :Binding to target 3 :Washing of non binding Ca 2+ 4 :Ca 2+ enables catalysis 4 :Ca 2+ enables catalysis Ca 5 :DNaseI elutes rest 4 After two rounds of panning several promising clones were isolated promising clones were isolated DNaseI 5

Summary • Conceived idea of a universal restriction enzyme. Conceived idea of a universal restriction enzyme. • Modeled 3D structure and the reaction kinetics. • Modularly cloned according to RFC25 (Freiburg fusion standard) d l l l d di C2 ( ib f i d d) • 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 were interviewed i i d • >35 parts were submitted and well documented. Scar issue for RFC25 was resolved within the BioBrick registry. Previous parts were updated.

Acknowledgements Instructors Instructors Support & Instrumentation 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