2013 EPFL Team Project
The 2013 EPFL iGEM Team 2
Problem Colorectal cancer: 3 rd most diagnosed Bacterial infection cancer in the world Crohn’s disease Parasites 3 3
Modular Targeted Delivery 4
Interdisciplinary Approach PHARMACOLOGY PHARMACOLOGY SYNTHETIC BIOLOGY SYNTHETIC BIOLOGY TAXI.COLI TAXI.COLI CHEMISTRY CHEMISTRY MATERIAL SCIENCE MATERIAL SCIENCE DISEASE DISEASE TREATMENT TREATMENT 5
Taxi.Coli 6
Taxi.Coli 7
http://www.youtube.com/watch?v=2oGyBahBHuI 8
Project Cell Surface Cell Surface Cell Surface Cell Surface Sensing Sensing Sensing Sensing Effector Effector Effector Effector Nanoparticles Nanoparticles Nanoparticles Nanoparticles Display Display Display Display 9
Cell Surface Sensing Effector Nanoparticles Display 10
Sensing Cell Nano Sensing Effector Surface particles D isplay • 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 11
Cloning strategy Cell Nano Sensing Effector Surface particles D isplay BBa_I746908 Constitutive Promoter Cad Promoter Hya Promoter Positive Control BBa_K1111005 BBa_K1111002 BBa_K1111004 12
Characterization: Microscopy Cell Nano Sensing Effector Surface particles D isplay Hya Promoter pH = 7.0 pH = 5.5 pH = 8.5 Cad Promoter pH = 7.0 pH = 8.5 pH = 5.5 Constitutive Promoter pH = 8.5 pH = 5.5 pH = 7.0 13
Plate Reader Cell Nano Sensing Effector Surface particles D isplay pH: 5.5 pH: 6.5 2 2 Hya-Promoter Hya-Promoter 1.5 1.5 OD 600 OD 600 Cad-Promoter 1 Cad-Promoter 1 0.5 0.5 Constitutive Constitutive Promoter Promoter 0 0 0 5 10 0 5 10 # of measurements [30min] # of measurements [30min] pH: 7 pH: 8.5 2.5 2 2 Hya-Promoter Hya-Promoter 1.5 1.5 OD 600 OD 600 Cad-Promoter 1 Cad-Promoter 1 Constitutive 0.5 0.5 Constitutive Promoter Promoter 0 0 0 5 10 0 5 10 # of measurements [30min] # of measurements [30min] 14
Achievements • Extraction of Genomic DNA • Promoter Insertion • 100% Sequencing Match • Positive Control Creation • Proof of Functionality Next Steps • Improve characterization • Develop new constructs 15
Cell Surface Sensing Effector Nanoparticles Display 16
Effector Cell Nano Sensing Effector Surface particles D isplay • Enzymes degrade gelatin nanoparticles • Two Different Enzymes – Gelatinase E (GelE) from E.faecalis – Matrix Metalloprotease 2 (MMP2) from H.sapiens 17
Effector Cell Nano Sensing Effector Surface particles D isplay His-Tag BBa_K1111009 BBa_I746908 His-Tag BBa_K1111007 18
Achievements • Extraction of Genomic DNA • Protein cDNA insertion into plasmid Next Steps • Protein Purification • Nanoparticles Degradation with the enzyme • Sensor and effector cloning into the same plasmid 19
Cell Surface Sensing Effector Nanoparticles Display 20
Streptavidin Display Cell Nano Sensing Effector Surface particles D isplay • Streptavidin fused to an Outer Membrane Protein • Chose Ice Nucleation Protein from Pseudomonas syringae 21
Cloning INP_Streptavidin Protein Cell Nano Sensing Effector Surface particles D isplay Streptavidin Dead Streptavidin Alive Streptavidin from Registry BBa_K1111012 BBa_K1111013 BBa_K1111014 22
ImmunoFluorescent Staining Cell Nano Sensing Effector Surface particles D isplay Negative Control INP-Streptavidin-Alive Composite image Composite image FITC conjugated Anti-streptvidin Ab, 100x FITC conjugated Anti-streptavidin Ab, 100x No Fluorescence → No AnDbody Binding 23
Biotin Staining Cell Nano Sensing Effector Surface particles D isplay Zoom-In INP-Streptavidin-Alive Composite images Composite image FITC conjugated Biotin, 100x 24
Biotin Staining Cell Nano Sensing Effector Surface particles D isplay Negative Control INP-Streptavidin-Alive Composite image Composite image FITC conjugated Biotin, 100x FITC conjugated Biotin, 100x 25
Edinburgh 2011 BBa_K523013 Cell Nano Sensing Effector Surface particles D isplay Control: BBa_K523013 YFP in the transformed cells Cytoplasm More Characterization Needed 26
Improved Cell Characterization Nano Sensing Effector Surface particles D isplay Confocal Microscopy: BBa_K523013 transformed cells Zoom 27
Improved Cell Characterization Nano Sensing Effector Surface particles D isplay Inverted Microscopy BBa_K523013 transformed cells Merge image of A & B YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5 Proves Surface Localisation 28
Improved Cell Characterization Nano Sensing Effector Surface particles D isplay Inverted Microscopy Negative Control WT E.coli, neg ctl, Merge image 29
INP-Streptavidin Expression Cell Nano Sensing Effector Surface particles D isplay Western Blot of Cell Pellets containing INP-Streptavidin • ~ 50 kDa expected • Cell pellets • FITC conjugated Anti- Streptavidin Antibody • Typhoon Scanner 30
New Cloning Cell Nano Sensing Effector Surface particles D isplay Added EYFP 31
ImmunoFluorescent Staining Cell Nano Sensing Effector Surface particles D isplay INP_YFP_Streptavidin YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5 Merge image of A & B INP_Streptavidin_YFP Merge image of A & B YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5 32
ImmunoFluorescent Staining Controls Cell Nano Sensing Effector Surface particles D isplay Positive Control: BBa_K523013 YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5 Merge image of A & B Negative Control: Competent Cells Merge image of A & B 33 YFP Fluorescence, 100x , FITC filter Anti-YFP Ab fluorescence , 100x, Cy5
Achievements • BBa_K523013 Characterization • Fusion Protein Cloning • Sequencing Confirmation • ImmunoFluorescent Assays • Biotin Staining Assays • Fusion protein IYS & ISY 34
Cell Surface Sensing Effector Nanoparticles Display 35
Nanoparticles Cell Nano Sensing Effector Surface particles D isplay 36
Making & Characterizing Nanoparticles Cell Nano Sensing Effector Surface particles D isplay Two Step Desolvation Method Size Distribution by Intensity Average size ~ 160 nm DLS: Dynamic Light Scattering 37
Two T ypes of Cargo Cell Nano Sensing Effector Surface particles D isplay Internal Loading External Labeling Inverted Microscopy Inverted Microscopy Nanoparticles containing CY5 attached to nanoparticles fluorescently labeled dextran 38
Biotinylation Cell Nano Sensing Effector Surface particles D isplay • Chemical Reaction using NHS-ester Biotinylated Gelatin Nanoparticles Biotin Nanoparticle 39
Biotinylated Nanoparticles Cell Nano Sensing Effector Surface particles D isplay ELISA-like Assay HRP-conjugated Avidin (commonly used in ELISA) Biotin Gelatin Nanoparticle Plate’s Well 40
Microfluidics: MITOMI Experiment Cell Nano Sensing Effector Surface particles D isplay Digestion of nanoparticles by MMP2 Before wash After wash After MMP2 flow 41
Attaching Nanoparticles to E.coli Cell Nano Sensing Effector Surface particles D isplay Nanoparticles Biotin Zoom-In + E.coli chemically linked to streptavidin Inverted Microscopy 42
Achievements • Successful Creation and Characterization of 5 batches of nanoparticles: Naked Biotinylated CY5-labeled biotinylated FITC-Dextran loaded Biotinylated FITC-Dextran loaded 43
Outreach Women&Engineering’s Educational Kit day 44
Educational kit EDUCATION & SAFETY FUN & SHARING « 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 45
Women&Engineering’s day 46
Design and Documentation Characterization of and Registry entry Amazing Summer of new Biobricks new Biobricks Functionality of Improved Careful scientific Characterization of BBa_K1111005 approach proved BBa_K523013 Exceptional Fantastic Human Autonomous microscopy assays Practice Wetlab work Autonomous Nanoparticles Organization 47
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 48
1 / 1 Questions? 49
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