Team: The Students Maria K. Shiva Ingrid Fadum Ellen Stormo - - PowerPoint PPT Presentation

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Team: The Students Maria K. Shiva Ingrid Fadum Ellen Stormo - - PowerPoint PPT Presentation

Jan 12, 2023 316 likes •626 views

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

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Team: The Students

Maria K. Andersen

  • Biotechnology/

Molecular medicine

  • Master student

Shiva Moghaddam

  • Molecular

medicine

  • Master student

Ellen Stormo

  • Biotechnology
  • Master student

Patricia Adl

  • Medical

technology/ Biophysics

  • Master student

Ingrid Fadum Kjønstad

  • Biochemistry/

Molecular medicine

  • Master student
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VesiColi

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Outer Membrane Vesicles – OMVs

Nanosized Quorum sensing Pathogenesis Transport of proteins Development of biofilm

Why not engineer OMVs to be drug delivery vehicles?

Kulp, A. and M. J. Kuehn (2010). "Biological functions and biogenesis of secreted bacterial outer membrane vesicles." Annu Rev Microbiol 64: 163-184 ,[2] Kesty, N. C., K. M. Mason, et al. (2004). "Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells." EMBO J 23(23): 4538-4549

Figure: Gold labeled vesicles from enterogenic E.coli that binds and internalizes in HT29 cancer cells [2]

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Overview

  • Introduce fluorescent proteins into OMVs
  • Make OMVs stable in the blood stream
  • Regulate what enters the vesicles
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How to direct proteins into the vesicles?

Twin-Arginine Signal Pathway Tat signal peptide

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Our construct in a plasmid

Tat signal sequence Coding sequence

Will direct the protein product to the periplasm

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Transport through the Tat transport pathway

Budding off!

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Vesicle Isolation

Sample pellet Analyze vesicle sample

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What to put in the vesicles?

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): 47-53.

BioBrick: BBa_K1082001

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Challenges in using OMVs as drug delivery vehicles

Stable in the blood stream Targeting specific cells Contain a drug

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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): 2469-2476

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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_K1082002)

"The Pm/xylS expression system." Retrieved 4.10.2013, from http://www.vectronbiosolutions.com/info.php?id=13.

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  • We succesfully made a tat-GFP-RFP

gene construct

  • Sequence confirmed!
  • Red fluorescence, but no green

fluorescence

  • No detectable quantity in OMVs

EITHER OR

Results: FP dimers

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We succesfully made a tat-Protein G construct tat-Protein G is expressed in E.coli

No detectable quantity in OMVs Inconclusive

Results: Protein G

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The Pm/Xyls promoter system

GFP fluorescence

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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!
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Human Practices

Researchers Night

  • 1100 High School students

Schrödingers Katt

  • TV-program about Science
  • Aired in january
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BioBricks

BioBrick Type Description Length (bp)

Part:BBa_K1082001 Coding Tat_GFP_RFP 1711 Part:BBa_K1082002 Regulatory Pm/Xyls promotor system 1760

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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

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Thank you for your time We had fun!

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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): 13183-13192.

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

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Red Fluorescence

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Excitation scan of OMVs

Tat_GFP_RFP construct Wild type