2013 EPFL Team Project2013.igem.org/files/presentation/EPF_Lausanne_Championship.pdf · Natalie...
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2013 EPFL Team Project
Transcript of 2013 EPFL Team Project2013.igem.org/files/presentation/EPF_Lausanne_Championship.pdf · Natalie...
2013 EPFL Team Project
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The 2013 EPFL iGEM Team
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.COLITAXI.COLI
SYNTHETIC BIOLOGYSYNTHETIC BIOLOGY
CHEMISTRYCHEMISTRY
DISEASE
TREATMENT
DISEASE
TREATMENT
MATERIAL SCIENCEMATERIAL SCIENCE
PHARMACOLOGYPHARMACOLOGY
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Taxi.Coli
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Taxi.Coli
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http://www.youtube.com/watch?v=2oGyBahBHuI
Project
Sensing EffectorCell Surface
DisplayNanoparticlesSensing Effector
Cell Surface
DisplayNanoparticlesSensing Effector
Cell Surface
DisplayNanoparticlesSensing Effector
Cell Surface
DisplayNanoparticles
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Sensing EffectorCell Surface
DisplayNanoparticles
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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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Constitutive PromoterPositive Control
Cloning strategy
BBa_I746908
Sensing Effector
Cell
Surface
Display
Nano
particles
Cad PromoterHya 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 ReaderSensing Effector
Cell
Surface
Display
Nano
particles
0
0.5
1
1.5
2
0 5 10
OD
60
0
# of measurements [30min]
pH: 5.5
Hya-Promoter
Cad-Promoter
Constitutive
Promoter0
0.5
1
1.5
2
0 5 10
OD
60
0
# of measurements [30min]
pH: 6.5
Hya-Promoter
Cad-Promoter
Constitutive
Promoter
0
0.5
1
1.5
2
2.5
0 5 10
OD
60
0
# of measurements [30min]
pH: 7
Hya-Promoter
Cad-Promoter
Constitutive
Promoter0
0.5
1
1.5
2
0 5 10
OD
60
0
# 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
Sensing EffectorCell Surface
DisplayNanoparticles
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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 EffectorCell Surface
DisplayNanoparticles
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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_K1111013BBa_K1111012 BBa_K1111014
Streptavidin DeadStreptavidin Alive
Sensing Effector
Cell
Surface
Display
Nano
particles
ImmunoFluorescent
Staining
Negative ControlComposite image
FITC conjugated Anti-streptvidin Ab, 100x FITC conjugated Anti-streptavidin Ab, 100x
INP-Streptavidin-AliveComposite image
No Fluorescence
→ No AnDbody Binding
Sensing Effector
Cell
Surface
Display
Nano
particles
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FITC conjugated Biotin, 100x
Biotin StainingSensing Effector
Cell
Surface
Display
Nano
particles
Zoom-InComposite images
INP-Streptavidin-AliveComposite image
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FITC conjugated Biotin, 100x FITC conjugated Biotin, 100x
Biotin StainingSensing Effector
Cell
Surface
Display
Nano
particles
Negative ControlComposite image
INP-Streptavidin-AliveComposite 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
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 & BYFP Fluorescence, 100x , FITC filter
Anti-YFP Ab fluorescence , 100x, Cy5 Merge image of A & BYFP Fluorescence, 100x , FITC filter
Achievements
• BBa_K523013 Characterization
• Fusion Protein Cloning
• Sequencing Confirmation
• ImmunoFluorescent Assays
• Biotin Staining Assays
• Fusion protein IYS & ISY
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Sensing EffectorCell Surface
DisplayNanoparticles
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NanoparticlesSensing 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
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
Attaching Nanoparticles
to E.coli
Nanoparticles
Biotin
E.coli chemically
linked to streptavidin
+
Sensing Effector
Cell
Surface
Display
Nano
particles
Zoom-In
Inverted Microscopy
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Achievements
• Successful Creation and Characterization of 5 batches
of nanoparticles:
Naked
Biotinylated
CY5-labeled biotinylated
FITC-Dextran loaded
Biotinylated FITC-Dextran loaded
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Outreach
Women&Engineering’s
dayEducational 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
Amazing Summer
Documentation
and Registry entry of new Biobricks
Design and
Characterization of
new Biobricks
ImprovedCharacterization of
BBa_K523013
AutonomousOrganization
Careful scientificapproach
Exceptionalmicroscopy assays
Fantastic HumanPractice
AutonomousWetlab work
Functionality of BBa_K1111005
proved
Nanoparticles
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ThanksEPFL 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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Questions?
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