2013 EPFL Team Project The 2013 EPFL iGEM Team 2 Problem - - PowerPoint PPT Presentation

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2013 EPFL Team Project The 2013 EPFL iGEM Team 2 Problem - - PowerPoint PPT Presentation

Oct 22, 2022 245 likes •756 views

2013 EPFL Team Project The 2013 EPFL iGEM Team 2 Problem Colorectal cancer: 3 rd most diagnosed Bacterial infection cancer in the world Crohns disease Parasites 3 3 Modular Targeted Delivery 4 Interdisciplinary Approach

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2013 EPFL Team Project

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The 2013 EPFL iGEM Team

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Problem

Bacterial infection Parasites Colorectal cancer: 3rd most diagnosed cancer in the world Crohn’s disease

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Modular Targeted Delivery

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Interdisciplinary Approach

TAXI.COLI TAXI.COLI

SYNTHETIC BIOLOGY SYNTHETIC BIOLOGY CHEMISTRY CHEMISTRY DISEASE TREATMENT DISEASE TREATMENT MATERIAL SCIENCE MATERIAL SCIENCE PHARMACOLOGY PHARMACOLOGY

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Taxi.Coli

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Taxi.Coli

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http://www.youtube.com/watch?v=2oGyBahBHuI

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SLIDE 9

Project

Sensing Effector Cell Surface Display Nanoparticles Sensing Effector Cell Surface Display Nanoparticles Sensing Effector Cell Surface Display Nanoparticles Sensing Effector Cell Surface Display Nanoparticles

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SLIDE 10

Sensing Effector Cell Surface Display Nanoparticles

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Sensing

  • pH sensitive promoters:

– Hya-promoter, isolated from the E.coli K-12 MG1655 – Cad-promoter, isolated from the E.coli K-12 MG1655

  • Positive control: constitutive promoter

Sensing Effector Cell Surface

Display

Nano particles

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SLIDE 12

Constitutive Promoter

Positive Control

Cloning strategy

BBa_I746908

Sensing Effector Cell Surface

Display

Nano particles

Cad Promoter Hya Promoter

BBa_K1111002 BBa_K1111004 BBa_K1111005

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Characterization: Microscopy

pH = 5.5 pH = 7.0 pH = 8.5 pH = 5.5 pH = 7.0 pH = 8.5 pH = 5.5 pH = 7.0 pH = 8.5

Sensing Effector Cell Surface

Display

Nano particles

Constitutive Promoter Cad Promoter Hya Promoter

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Plate Reader

Sensing Effector Cell Surface

Display

Nano particles

0.5 1 1.5 2 5 10 OD600 # of measurements [30min]

pH: 5.5

Hya-Promoter Cad-Promoter Constitutive Promoter 0.5 1 1.5 2 5 10 OD600 # of measurements [30min]

pH: 6.5

Hya-Promoter Cad-Promoter Constitutive Promoter 0.5 1 1.5 2 2.5 5 10 OD600 # of measurements [30min]

pH: 7

Hya-Promoter Cad-Promoter Constitutive Promoter 0.5 1 1.5 2 5 10 OD600 # of measurements [30min]

pH: 8.5

Hya-Promoter Cad-Promoter Constitutive Promoter

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Achievements

  • Extraction of Genomic DNA
  • Promoter Insertion
  • 100% Sequencing Match
  • Positive Control Creation
  • Proof of Functionality

Next Steps

  • Improve characterization
  • Develop new constructs
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SLIDE 16

Sensing Effector Cell Surface Display Nanoparticles

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Effector

  • Enzymes degrade gelatin nanoparticles
  • Two Different Enzymes

– Gelatinase E (GelE) from E.faecalis – Matrix Metalloprotease 2 (MMP2) from H.sapiens

Sensing Effector Cell Surface

Display

Nano particles

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Effector

BBa_K1111009

Sensing Effector Cell Surface

Display

Nano particles

BBa_I746908 BBa_K1111007

His-Tag His-Tag

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Achievements

  • Extraction of Genomic DNA
  • Protein cDNA insertion into plasmid
  • Protein Purification
  • Nanoparticles Degradation with the enzyme
  • Sensor and effector cloning into the same

plasmid

Next Steps

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Sensing Effector Cell Surface Display Nanoparticles

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Streptavidin Display

  • Streptavidin fused to an Outer Membrane

Protein

  • Chose Ice Nucleation Protein from

Pseudomonas syringae

Sensing Effector Cell Surface

Display

Nano particles

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Cloning INP_Streptavidin Protein

Streptavidin from Registry

BBa_K1111013 BBa_K1111012 BBa_K1111014

Streptavidin Dead Streptavidin Alive

Sensing Effector Cell Surface

Display

Nano particles

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SLIDE 23

ImmunoFluorescent Staining

Negative Control

Composite image

FITC conjugated Anti-streptvidin Ab, 100x FITC conjugated Anti-streptavidin Ab, 100x

INP-Streptavidin-Alive

Composite image

No Fluorescence → No AnDbody Binding

Sensing Effector Cell Surface

Display

Nano particles

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FITC conjugated Biotin, 100x

Biotin Staining

Sensing Effector Cell Surface

Display

Nano particles

Zoom-In

Composite images

INP-Streptavidin-Alive

Composite image

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SLIDE 25

FITC conjugated Biotin, 100x FITC conjugated Biotin, 100x

Biotin Staining

Sensing Effector Cell Surface

Display

Nano particles

Negative Control

Composite image

INP-Streptavidin-Alive

Composite image

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Edinburgh 2011 BBa_K523013

Control: YFP in the Cytoplasm BBa_K523013 transformed cells

More Characterization Needed

Sensing Effector Cell Surface

Display

Nano particles

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Improved Characterization

Confocal Microscopy: BBa_K523013 transformed cells

Zoom

Sensing Effector Cell Surface

Display

Nano particles

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Improved Characterization

YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5

Merge image of A & B

Inverted Microscopy BBa_K523013 transformed cells Proves Surface Localisation

Sensing Effector Cell Surface

Display

Nano particles

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WT E.coli, neg ctl, Merge image

Improved Characterization

Sensing Effector Cell Surface

Display

Nano particles

Inverted Microscopy Negative Control

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INP-Streptavidin Expression

Western Blot of Cell Pellets containing INP-Streptavidin

  • ~ 50 kDa expected
  • Cell pellets
  • FITC conjugated

Anti-Streptavidin Antibody

  • Typhoon Scanner

Sensing Effector Cell Surface

Display

Nano particles

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New Cloning

Added EYFP

Sensing Effector Cell Surface

Display

Nano particles

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ImmunoFluorescent Staining

Sensing Effector Cell Surface

Display

Nano particles

Anti-YFP Ab fluorescence , 100x, Cy5 Anti-YFP Ab fluorescence , 100x, Cy5

Merge image of A & B Merge image of A & B

YFP Fluorescence, 100x , FITC filter YFP Fluorescence, 100x , FITC filter

INP_YFP_Streptavidin INP_Streptavidin_YFP

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ImmunoFluorescent Staining Controls

Sensing Effector Cell Surface

Display

Nano particles

Positive Control: BBa_K523013 Negative Control: Competent Cells

Anti-YFP Ab fluorescence , 100x, Cy5

Merge image of A & B

YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5

Merge image of A & B

YFP Fluorescence, 100x , FITC filter

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Achievements

  • BBa_K523013 Characterization
  • Fusion Protein Cloning
  • Sequencing Confirmation
  • ImmunoFluorescent Assays
  • Biotin Staining Assays
  • Fusion protein IYS & ISY

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SLIDE 35

Sensing Effector Cell Surface Display Nanoparticles

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Nanoparticles

Sensing Effector Cell Surface

Display

Nano particles

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Making & Characterizing Nanoparticles

Two Step Desolvation Method DLS: Dynamic Light Scattering

Sensing Effector Cell Surface

Display

Nano particles

Average size ~ 160 nm

Size Distribution by Intensity

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Two Types of Cargo

Nanoparticles containing fluorescently labeled dextran

Internal Loading External Labeling

CY5 attached to nanoparticles

Sensing Effector Cell Surface

Display

Nano particles

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Inverted Microscopy Inverted Microscopy

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Biotinylation

  • Chemical Reaction using NHS-ester

Sensing Effector Cell Surface

Display

Nano particles

Biotin Gelatin Nanoparticle Biotinylated Nanoparticles

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Biotinylated Nanoparticles

ELISA-like Assay

Sensing Effector Cell Surface

Display

Nano particles

HRP-conjugated Avidin (commonly used in ELISA) Biotin Gelatin Nanoparticle Plate’s Well

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Microfluidics: MITOMI Experiment

Digestion of nanoparticles by MMP2

Sensing Effector Cell Surface

Display

Nano particles

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Before wash After wash After MMP2 flow

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Attaching Nanoparticles to E.coli

Nanoparticles Biotin E.coli chemically linked to streptavidin

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Sensing Effector Cell Surface

Display

Nano particles

Zoom-In Inverted Microscopy

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Achievements

  • Successful Creation and Characterization of 5 batches
  • f nanoparticles:

Naked Biotinylated CY5-labeled biotinylated FITC-Dextran loaded Biotinylated FITC-Dextran loaded

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Outreach

Women&Engineering’s day Educational Kit

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Educational kit

« Education that is challenging and fun is really at the heart of what we do- and it sounds like what you do

  • too. » 
Natalie Kuldell: President of Biobuilder

educational foundation

FUN & SHARING EDUCATION & SAFETY

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Women&Engineering’s day

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Amazing Summer Documentation and Registry entry

  • f new Biobricks

Design and Characterization of new Biobricks Improved Characterization of BBa_K523013 Autonomous Organization Careful scientific approach Exceptional microscopy assays Fantastic Human Practice Autonomous Wetlab work Functionality of BBa_K1111005 proved Nanoparticles

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Thanks

EPFL School of Engineering EPFL School of Life Sciences Our Sponsors The Biobuilder foundation and Natalie Kuldell The EPFL Equal Opportunities Office and Farnaz Moser

  • Dr. Daniel Bonner, Dr. David Scott Wilson and Alexandre De Titta

Thierry Laroche

  • Prof. Eric Allémann
  • Prof. Matthias Lutolf and Prof. McKinney

Michael Fairhead Nicole Paduwat and Stéphane Karlen Sabrina Leuenberger Sander Kromwijk

  • Dr. Sacha Sidjanski

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SLIDE 49

Questions?

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