Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

iGEM Slovenia @ 6th CeBiTec conference

582 views

Published on

Published in: Technology
  • Be the first to comment

iGEM Slovenia @ 6th CeBiTec conference

  1. 1. Slovenian Projects Jernej Turnšek Department of Biotechnology Follow me on Twitter: @SynEnthu6th CeBiTec Symposium Bielefeld, July 20th, 2011
  2. 2. Agenda 1) Slovenian iGEM timeline 2) nanoBRICKS[pro] – 2009 project 3) DNA coding beyond triplets – 2010 project 4) Slovenian iGEM teams 2006 – 2010 5) Track record6th CeBiTec Symposium Bielefeld, July 20th, 2011
  3. 3. Slovenian iGEM timeline 2006 – Engineered Human Cells – SAY NO TO SEPSIS 2007 – virotrap® – A Synthetic Biology Approach Against HIV 2008 – Immunobricks 2009  nanoBRICKS[pro] 2010  DNA coding beyond triplets6th CeBiTec Symposium Bielefeld, July 20th, 2011
  4. 4. [aim]  create self-assembling polypeptide nanostructures Regulated nanomaterial assembly Only 0.22um PVDF 0.22um PVDF Filter Filter with protein membrane PVDF filter PVDF filter Protein Polypeptide membranes with 0.22 with 0.22 membrane um pores um pores Coiled-coil-based nanostructure assembly6th CeBiTec Symposium Bielefeld, July 20th, 2011
  5. 5. [strategies for polypeptide self-assembly] BUILDING BLOCKS OLIGOMERISATION DOMAINS COILED-COIL SEGMENTS (dimer, trimer, tetramer, pentamer, hexamer...) (as rigid rods)  2-7 intertwined α-helices  8% of natural proteins  helices in (anti)parallel orientation functional self-assembling element6th CeBiTec Symposium Bielefeld, July 20th, 2011
  6. 6. [regulated nanomaterial assembly] GyrB assembly with coumermycin disassembly with novobiocin CutA1 Potential applications for drug delivery and controlled release. 200 nm TEM image6th CeBiTec Symposium Bielefeld, July 20th, 2011
  7. 7. [polypeptide membranes] p53 APH, APH1 or BCR - size defined mainly by the length of the coiled-coil segment - residues at the surface of CC segment define its chemical properties Potential applications for filtration and separation devices, catalysis.6th CeBiTec Symposium Bielefeld, July 20th, 2011
  8. 8. [coiled-coil-based nanostructure assembly] - 2 coiled-coils  only 1D fibrils - 3 coiled-coils  2D and 3D nano-objects Parallel homodimeric and two heterodimeric segments a b a’ polypeptide nanocube polypeptide network6th CeBiTec Symposium Bielefeld, July 20th, 2011
  9. 9. DNA coding beyond triplets iGEM 2010 Team Slovenia
  10. 10. Rethinking the central biological dogma6th CeBiTec Symposium Bielefeld, July 20th, 2011
  11. 11. DNA guides the assembly of functional proteins 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) Functional proteins perform sequential operations.6th CeBiTec Symposium Bielefeld, July 20th, 2011
  12. 12. Selection of DNA binding domains  helix-turn-helix, leucine zipper, zinc finger  modular design  >700 characterised ZNFs already available in ZiFDB  262.144 possible binding sites for nonameres (49)  longer recognition sequence  more specific binding6th CeBiTec Symposium Bielefeld, July 20th, 2011
  13. 13. Characterisation of ZNFs Gli1, HIVC, Zif268, PBSII, Jazz, Blues  cloned  expressed  purified  characterized Surface plasmon resonance (SPR) EMSA binding dissociation Resp. Diff. [RU] time [s] Time [s] All isolated ZNFs bind to their target DNA.6th CeBiTec Symposium Bielefeld, July 20th, 2011
  14. 14. ZNF binding to target DNA in vivo ZNFs act as artificial repressors.6th CeBiTec Symposium Bielefeld, July 20th, 2011
  15. 15. Assembly of multiple DNA binding domains6th CeBiTec Symposium Bielefeld, July 20th, 2011
  16. 16. Resveratrol biosynthesis  2-step biosynthetic pathway 4CL: 4-coumarate:CoA ligase STS: stilbene synthase  antioxidant  French paradox6th CeBiTec Symposium Bielefeld, July 20th, 2011
  17. 17. DNA-guided resveratrol biosynthesis a: Zif268 + E1: 4CL = Zif268::4CL fusion b: PBSII + E2: STS = PBSII::STS fusion D 50 40 4CL::STS 30 * mAU 20  length of the DNA program: 16x scaffold with 2, 4 or 8bp spacer 10  compared to direct fusion 4CL::STS and no DNA program 0 0 5 10 15 20 25 30 35 Minutes D 50 50 40 4CL::STS 40 2bp spacer 30 * 30 mAU mAU 20 20 * 10 10 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Minutes Minutes 506th CeBiTec Symposium Bielefeld, July 20th, 2011
  18. 18. Results summary 8 15 B C resveratrol (mg/l) resveratrol (mg/l) 6 10 4 5 2 0 0 2h 6h h 27 m r r er on 4b ace 8b ace ra ac 4CL::STS fusion 4bp spacer si og sp sp sp fu pr TS p p p no program 8bp spacer 2b no :S L: 2bp spacer 4C6th CeBiTec Symposium Bielefeld, July 20th, 2011
  19. 19. Conclusions • we invented a new type of ordered protein assembly based on a DNA program 8 • exciting potentials for biosynthesis B resveratrol (mg/l) 6 • yield improvement 4 • chanelling 2 0 m r er er on 4b ace ra ac ac si og sp sp sp fu pr TS p p p 2b 8b no :S L: • 151 BioBricks submitted to the Registry: 4C universal DNA binding domains testing device, split GFPs, zinc fingers...6th CeBiTec Symposium Bielefeld, July 20th, 2011
  20. 20. Future perspectives • complex (and multiple) biosynthetic pathways with several reactions (e.g. drugs, biofuels…) • in vitro scaffold for multienzyme reactions Beyond biosynthesis: • pattern recognition • information processing (using protein kinases, proteases etc.) Keasling et al., 2003, Nature Biotechnology6th CeBiTec Symposium Bielefeld, July 20th, 2011
  21. 21. Human practice – make it understandable6th CeBiTec Symposium Bielefeld, July 20th, 2011
  22. 22. Slovenian iGEM teams 2006-2010 2006 2007 2008 2009 2010 Monika Ciglič Marko Bitenc Eva Čeh Sabina Božič Jernej Turnšek Ota Fekonja Peter Cimermančič Vid Kočar Nika Debeljak Nejc Tomšič Jernej Kovač Rok Gaber Katja Kolar Tibor Doles Tjaša Stošicki Alja Oblak Saša Jereb Ana Lasič Urška Jelerčič Tina Ilc Jelka Pohar Katja Kolar Jan Lonzarić Anja Lukan Tina Lebar Matej Skočaj Anja Korenčič Jerneja Mori Špela Miklavič Matej Žnidarič Rok Tkavc Andrej Ondračka Anže Smole Marko Verce Mattia Petroni Jure Bordon Rok Pustoslemšek Rok ČrešnovarMentorsMojca Benčina, Monika Avbelj, Karolina Ivičak, Nina Pirher, Gabriela Panter, Mateja Manček Keber, Iva Hafner Bratkovič, HelenaGradišar, Ota Fekonja, Jelka Pohar, Rok Gaber, Tomaž Koprivnjak, Jerneja Mori, Irena Vovk (KI), Marko Dolinar (FKKT), Simon Horvat(BF, KI), Gregor Anderluh (KI, BF), Vesna Hodnik (BF), Miha Mraz, Miha Moškon, Nikolaj Zimic (FRI), Roman Jerala (KI, FKKT, En-FIST)6th CeBiTec Symposium Bielefeld, July 20th, 2011
  23. 23. Sponsors6th CeBiTec Symposium Bielefeld, July 20th, 2011
  24. 24. Track record 2006 – 2010  Gold medal: 5x  Finalists: 4x  Best in Area: 4x  Grand Prize: 3x (2006, 2008, 2010)6th CeBiTec Symposium Bielefeld, July 20th, 2011
  25. 25. Questions & Answers
  26. 26. [comparison of DNA and polypeptides as nanostructure building blocks] DNA polypeptides contains 4 nucleotides with similar contain 20 AA with different chemical properties [-] properties [+] can fold into defined 3D structures can fold into defined 3D structure [+] [+/-] build structures and functional devices used to store information in nature [-] in nature [+] structure-encoding information is easy to program (W-C base pairs) [+] complex [-]6th CeBiTec Symposium Bielefeld, July 20th, 2011
  27. 27. Protein scaffold vs. DNA scaffold DNA scaffold Protein scaffold Spatial orientation Bundled Linear Order Unpredictable Highly predictable order Scaffold: binding Similar, well characterized Variations in strength domain interactions interactions Limited number of Large number of DNA Number of available protein dimerization binding domains is readily elements domains available available6th CeBiTec Symposium Bielefeld, July 20th, 2011
  28. 28. Role of the spacer length6th CeBiTec Symposium Bielefeld, July 20th, 2011
  29. 29. Confirmation of FRET Photobleaching of the acceptor molecule (YFP). Quantification of the FRET effect upon the addition of a DNA program. Four zinc fingers linked to funtional domains can bind simultaneously to neighbouring sites on DNA6th CeBiTec Symposium Bielefeld, July 20th, 2011

×