Synthetic Biologyannouncing the advancing technological revolution            Roman Jerala        Department of biotechnol...
The miracle of a Seed
DNA as the blueprint of life
Genome sequencing2001 Draft of thehuman genome       1000 genomes project initiated in 2008  60x more sequence data than c...
Information stored in the DNAHow much of DNA is on the world:•   4*1035 bp bacterial DNA (5% DNA)•   4*1034 bp plant DNA (...
Chemical synthesis of          oligonucleotides                 annealing                ligation                         ...
The assembly of a synthetic M. mycoides               genome                                             Assembled in yeas...
Metabolic pathways of a cell
The engineering approach of        synthetic biology• Application of engineering principles  into biological systems• Impo...
(Synthetic) Biology isnanotechnology that works.                               Drew Endyhttp://www.arn.org/mm/mm.htm
Synthetic biology as an          investigative tool                  “What I can not create, I do not                  und...
Področja uporabe sintezne biologije    Synthetic biology contributes        to solve problems in    •   Health/Medicine   ...
Synthetic biology to                  (re)design products  More efficient and  sustainable,  production:  -Drugs to treat ...
Complex biosynthetic pathways
Limitation of multienzyme biosynthetic pathways           Enzyme           Enzyme             3                4          ...
Scaffolding of biosynthetic pathway enzymes toenhance the biosynthetic rate   Enzyme     Enzyme      Enzyme     Enzyme    ...
Improved biosynthesis using DNA scaffold                           the order of DNA motifs along the                      ...
Improved biosynthesis using DNA scaffold                                               300% yield improvement             ...
Sintezna biologija za obnovljive vire energije   Renewable sources of energy               CO2                            ...
Logic-gated nanorobot for drug               delivery                               DNA origami forms a                   ...
Binding to cells depending on thecombination of surface-exposed markers
Nanostructures in nature Transcending the nature Can we bend molecules at will ?
Polypeptide origami•   Polypeptides fold into defined tertiary structures• Nature solved the design problem during hundred...
Design of complex 3D structures                                                   Deconstruct the designed               ...
Coiled-coil  interactions governing the specificity of coiled-coil formation are well understood                     coi...
Flexible linker between building elements    How can we build complex structures from rigid building blocks ?
Flexible linker between building elements       Each edge in the final structure is composed of a dimer
Design of a tetrahedron-forming polypeptide                                                   4 parallel dimers  APHP3BC...
Polypeptide production, isolation, assemblyProduction in E.coli                      Self-assembly                        ...
Detection of nano-tetrahedra                                HHHHHH           Negative staining          Nanogold 2 nm     ...
N- an C-terminal ends of the tetrahedral path coincide
“See things not as they are, but as they  might be”Robert OppenheimerWatermark in the synthetic genome of Mycoplasma labor...
Slovenian teams 2006-2010                     2007                      2008             2009                  20102006   ...
OBC | Synthetic biology announcing the coming technological revolution
OBC | Synthetic biology announcing the coming technological revolution
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OBC | Synthetic biology announcing the coming technological revolution

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Roman Jerala, National Institute of Chemistry, Ljubljana, Slovenia
Synthetic biology announcing the coming technological revolution

http://obc2012.outofthebox.si/

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OBC | Synthetic biology announcing the coming technological revolution

  1. 1. Synthetic Biologyannouncing the advancing technological revolution Roman Jerala Department of biotechnology National institute of chemistry, FKKT UL EN-FIST Centre of Excellence Ljubljana, Slovenia
  2. 2. The miracle of a Seed
  3. 3. DNA as the blueprint of life
  4. 4. Genome sequencing2001 Draft of thehuman genome 1000 genomes project initiated in 2008 60x more sequence data than combined previous 25 years
  5. 5. Information stored in the DNAHow much of DNA is on the world:• 4*1035 bp bacterial DNA (5% DNA)• 4*1034 bp plant DNA (1% DNA)• 1*1034 bp animal DNA (1% DNA)• 5*1030 bp viral DNA (50% DA)• 5*1035 bp of DNA on Earth~1 bit/nm3, much higher density than the existing information storage mediaGenome could be rewritten or modified
  6. 6. Chemical synthesis of oligonucleotides annealing ligation Insertion into plasmidIntroductioninto the cell
  7. 7. The assembly of a synthetic M. mycoides genome Assembled in yeast from 1078 overlapping DNA cassettes in three steps: 1. 1080 bp casettes from synthetic oligos for 10 kbp assemblies 2. 10 assemblies were combined into 100 kbp assemblies 3. 11 fragments were combined into the full chromosome D G Gibson et al. Science 2010;329:52-56
  8. 8. Metabolic pathways of a cell
  9. 9. The engineering approach of synthetic biology• Application of engineering principles into biological systems• Important engineering principles: – Modularity – Abstraction – Reliability – Predictability – Standardization Adriananoandro et el., Mol.Sys.Biol. 2006
  10. 10. (Synthetic) Biology isnanotechnology that works. Drew Endyhttp://www.arn.org/mm/mm.htm
  11. 11. Synthetic biology as an investigative tool “What I can not create, I do not understand!” How and why are natural systems constructed as they are ? (functional requirements or evolutionary coincidence) Do we understand the function of all components of a device/system?Richard Feynman
  12. 12. Področja uporabe sintezne biologije Synthetic biology contributes to solve problems in • Health/Medicine • Sustainable sources of energy • New materials & bionanomaterials • Information processing • Biosensors • Bioremediation...
  13. 13. Synthetic biology to (re)design products More efficient and sustainable, production: -Drugs to treat malaria -Biofuels -...http://keaslinglab.lbl.gov/application_areas/index.html
  14. 14. Complex biosynthetic pathways
  15. 15. Limitation of multienzyme biosynthetic pathways Enzyme Enzyme 3 4 Enzyme Enzyme 2 1
  16. 16. Scaffolding of biosynthetic pathway enzymes toenhance the biosynthetic rate Enzyme Enzyme Enzyme Enzyme 3 4 1 2
  17. 17. Improved biosynthesis using DNA scaffold 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)Bound enzymes perform sequential reactions
  18. 18. Improved biosynthesis using DNA scaffold 300% yield improvement 15 C resveratrol (mg/l) 10 4CL STS 5 0 2h 6h h 27 4CL::STS fusion 4bp spacer no program 8bp spacer 2bp spacer
  19. 19. Sintezna biologija za obnovljive vire energije Renewable sources of energy CO2 enzymes cellulose sugars CO2 Microbial conversion fuel
  20. 20. Logic-gated nanorobot for drug delivery DNA origami forms a hexagonal barrell split in half Complementary clamps close the barrell Binding sites inside the barrel can be loaded with cargo conjugated to ssDNADouglas et al., Science 2012
  21. 21. Binding to cells depending on thecombination of surface-exposed markers
  22. 22. Nanostructures in nature Transcending the nature Can we bend molecules at will ?
  23. 23. Polypeptide origami• Polypeptides fold into defined tertiary structures• Nature solved the design problem during hundreds of millionsof years of evolution
  24. 24. Design of complex 3D structures Deconstruct the designed structure into smaller rigid independently-folded building elements and Concatenate those elements into the self-assembling polypeptidehttp://bestbridge.net/Eu_en/the-iron-bridge.html
  25. 25. Coiled-coil  interactions governing the specificity of coiled-coil formation are well understood  coiled-coils are characterized by regular repeating unit of 7 amino acids heptad repeat (a-b-c-d-e-f-g)  stability and specificity is provided by residues at defined positions within the heptad repeat
  26. 26. Flexible linker between building elements  How can we build complex structures from rigid building blocks ?
  27. 27. Flexible linker between building elements Each edge in the final structure is composed of a dimer
  28. 28. Design of a tetrahedron-forming polypeptide 4 parallel dimers APHP3BCRGCNshAPHP7GCNshP4P5P8BCRP6 2 antiparallel dimersTET12 His6  flexible tetrapeptide linker
  29. 29. Polypeptide production, isolation, assemblyProduction in E.coli Self-assembly SDS-PAGE  Dialysis at low polypeptide concentrationProtein purificationAffinity chromatographyHPLC-RP
  30. 30. Detection of nano-tetrahedra HHHHHH Negative staining Nanogold 2 nm + Negative staining
  31. 31. N- an C-terminal ends of the tetrahedral path coincide
  32. 32. “See things not as they are, but as they might be”Robert OppenheimerWatermark in the synthetic genome of Mycoplasma laboratorium
  33. 33. Slovenian teams 2006-2010 2007 2008 2009 20102006 Marko Bitenc, BF Eva Čeh, BF Sabina Božič, FKKT Jernej TurnšekMonika Ciglič, BF Peter Cimermančič, FKKT Vid Kočar, FKKT Nika Debeljak, BF NejcOta Fekonja, BF Rok Gaber, BF Katja Kolar, FKKT Tibor Doles, BF TjašaJernej Kovač, FKKT Saša Jereb , FKKT Ana Lasič, MF Urška Jelerčič;FMF Tina IlcAlja Oblak, BF Katja Kolar, FKKT Jan Lonzarić, FKKTAnja Lukan, FKKT Tina LebarJelka Pohar, BF Anja Korenčič, FKKT Jerneja Mori, BF Špela Miklavič, BF Matej ŽnidaričMatej Skočaj, BF Andrej Ondračka, FKKT Anže Smole, BF Marko Verce, BF Mattia PetroniRok Tkavc, BF Jure Bordon Rok Pustoslemšek Rok ČrešnovarMentorsMojca Benčina, Monika Avbelj, Karolina Ivičak , Nina Pirher, Gabriela Panter, Mateja Manček Keber (KI), Marko Dolinar(FKKT), Simon Horvat (BF), Iva Hafner Bratkovič, Helena Gradišar, Ota Fekonja, Jelka Pohar, Rok Gaber, Tomaž Koprivnjak, Jerneja Mori, Irena Vovk, (KI), Gregor Anderluh, Vesna Hodnik (BF) , Miha Mraz, Miha Moškon, Nikolaj Zimic (FRI), Roman Jerala (KI, FKKT)

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