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DNA guided assembly line

Centre of Exellence. DNA guided assembly line. Rok Gaber Laboratory of Biotechnology. The 2nd International Symposium Vera Johanides Biotechnology in Croatia by 2020 11.5.2013. Many useful compounds are produced by natural microorganisms. …. Biomaterials .

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DNA guided assembly line

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  1. Centre ofExellence DNA guided assembly line Rok Gaber LaboratoryofBiotechnology The 2nd International Symposium Vera Johanides Biotechnology in Croatia by 2020 11.5.2013

  2. Manyusefulcompounds are producedbynaturalmicroorganisms … Biomaterials. Biologicallyactivesubstances. Pigments, Antioxidants, Foodsupplements. Biofuels. More.

  3. Inovative approach - scaffolding of biosynthetic pathway enzymes Enzyme 3 Enzyme 4 Enzyme 1 Enzyme 2

  4. Inovative approach - scaffolding of biosynthetic pathway enzymes Enzyme 3 Enzyme 4 Enzyme 1 Enzyme 2

  5. Inovativeapproach - scaffoldingofbiosyntheticpathwayenzymes • Directcovalentlinkeageofsuccesiveenzymes • Introductionof protein interactingdomains • Protein scaffolding • RNA scaffolding • DNA scaffolding

  6. Selectionof DNA bindingdomains • Sequencespecific • Highaffinity • Similarcharacteristics • Available in largenumber • Zinc fingerLeucinezipperHelix-turn-helix

  7. RNA polymerase RNA polymerase ZF Testing of selected Zinc Fingers Zincfingerexpression is notinduced. Zincfingerexpressionisinduced. X pBAD pBAD ZincFinger ZincFinger X X ZFBS ZFBS Reporter gene Reporter gene -35 -10 -35 -10

  8. TestingofZincFingers

  9. Proofofprinciple • CanZincFingerssimultaneouslybind to adjacentbindingsites?

  10. Proofofprinciple • Assay 1: • DNA enabledsplitfluorescent protein reconstitution. N-YFP C-YFP 2bp

  11. Proofofprinciple • Assay 1: • DNA enabledsplitfluorescent protein reconstitution.

  12. Model biosyntheticpathway – resveratrolbiosynthesis • Enzymesfromtwodifferentorganisms • Directcovalentlinkeageof 4CL and STS increasesproductionofresveratrol (Zhangetal. 2006). • Extendablebiosyntheticpathway TAL OMT

  13. ( ) ) ( ) ( 16X 2bp 4bp 8bp DesignofZincFinger – enzymefusions TAL OMT ( )

  14. Standards Finalresults trans-resveratrol p-coumaricacid pterostilbene 3-foldincreaseofresveratrolproduction in presenceof DNA scaffold. No program 2bp spacer

  15. Acknowledgementsandcontributions Roman Jerala iGEM 2010 teamSlovenia Jernej Turnšek Nejc Tomšič Tjaša Stošicki Tina Ilc Tina Lebar Matej Žnidarič MattiaPetroni Jure Bordon Rok Pustoslemšek Rok Črešnovar Miha Mraz Miha Moškon Nikolaj Zimic Monika Avbelj Jerneja Mori Jernej Turnšek Mojca Benčina Gregor Anderluh Irena Vovk Mateja Puklavec Tomaž Koprivnjak Rok Gaber Tina Lebar Vesna Hodnik Vesna Glavnik Breda Simonovska

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