Team: The Students. Maria K. Andersen. Shiva Moghaddam. Ingrid Fadum Kjønstad. Patricia Adl. Ellen Stormo

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2 Team: The Students Maria K. Andersen Shiva Moghaddam Ellen Stormo Patricia Adl Ingrid Fadum Kjønstad Biotechnology/ Molecular medicine Master student Molecular medicine Master student Biotechnology Master student Medical technology/ Biophysics Master student Biochemistry/ Molecular medicine Master student

3 VesiColi

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5 Outer Membrane Vesicles OMVs Nanosized Quorum sensing Pathogenesis Transport of proteins Development of biofilm Why not engineer OMVs to be drug delivery vehicles? Figure: Gold labeled vesicles from enterogenic E.coli that binds and internalizes in HT29 cancer cells [2] Kulp, A. and M. J. Kuehn (2010). "Biological functions and biogenesis of secreted bacterial outer membrane vesicles." Annu Rev Microbiol 64: ,[2] Kesty, N. C., K. M. Mason, et al. (2004). "Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells." EMBO J 23(23):

6 Overview Introduce fluorescent proteins into OMVs Make OMVs stable in the blood stream Regulate what enters the vesicles

7 How to direct proteins into the vesicles? Twin-Arginine Signal Pathway Tat signal peptide

8 Tat signal sequence Will direct the protein product to the periplasm Our construct in Coding sequence a plasmid

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10 Transport through the Tat transport pathway Budding off!

11 Vesicle Isolation Sample pellet Analyze vesicle sample

12 What to put in the vesicles? BioBrick: BBa_K Thomas, J. D., R. A. Daniel, et al. (2001). "Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli." Molecular Microbiology 39(1):

13 Challenges in using OMVs as drug delivery vehicles Contain a drug Targeting specific cells Stable in the blood stream

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15 Protein G Streptococcal species Transmembrane surface protein Binds human serum albumin (HSA) Can t be a BioBrick due to restriction sites Masking the vesicles from the immunesystem is crucial for their stability in the body as a drug carrier Egesten, A., I. M. Frick, et al. (2011). "Binding of albumin promotes bacterial survival at the epithelial surface." J Biol Chem 286(4):

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17 The Pm/Xyls promoter system Regulation of our vesicle system Protein G GFP-RFP Removed a XbaI restriction site to make it a biobrick (BBa_K ) "The Pm/xylS expression system." Retrieved , from

18 Results: FP dimers We succesfully made a tat-gfp-rfp gene construct Sequence confirmed! Red fluorescence, but no green fluorescence No detectable quantity in OMVs EITHER OR

19 Results: Protein G We succesfully made a tat-protein G construct tat-protein G is expressed in E.coli No detectable quantity in OMVs Inconclusive

20 The Pm/Xyls promoter system GFP fluorescence

21 Novel Approach: Engineered OMVs as drug velivery vehicles It is hard to make drug delivery vehicles synthetically Nanosized Safe (can t replicate) Naturally attack eukaryotic cells Has never been done before!

22 Human Practices Researchers Night 1100 High School students Schrödingers Katt TV-program about Science Aired in january

23 BioBricks BioBrick Type Description Length (bp) Part:BBa_K Coding Tat_GFP_RFP 1711 Part:BBa_K Regulatory Pm/Xyls promotor system 1760

24 Thank You Advisors! Eivind Almaas Professor, Systems Biology Rahmi Lale Postdoc, Molecular biology Gunvor Røkke Phd Student, Molecular biology Martin Hohmann- Marriott Associate Professor, Molcular Biology

25 Thank you for your time We had fun!

26 Linker sequence Repetitive sequence: (GGSGGS) 1-9 Fusion of protein domains via flexible peptide linkers - design proteins with new functions. peptide linker spatially separates the two proteins - functioning and folding of protein domains. Evers, T. H., E. M. van Dongen, et al. (2006). "Quantitative understanding of the energy transfer between fluorescent proteins connected via flexible peptide linkers." Biochemistry 45(44):

27 SDS-PAGE of OMVs from E.coli transformed with the tat_gfp_rfp construct

28 Red Fluorescence

29 Excitation scan of OMVs Tat_GFP_RFP construct Wild type