2013 biodesign EPFL project summary
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The EPFL team in Switzerland summarizes the motivation, direction of project and the prototypes accomplished during the spring of 2013
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2013 biodesign EPFL project summary
- 1. Final Presentation BIODESIGN FOR REAL WORLD 3rd of June 2013 Jasmina Rubattel Emilie Mussard Nicolas Krischer Romain Equey
- 2. Plan — Introduction — Aim — Motivation — Background researches — Coliform bacteria — Arsenic — Fluorescence — Legal framework — Decision — Design criteria — Prototype I: presentation and demo — Prototype II: presentation and demo — Data and analysis — Conclusions — Future directions
- 4. Aim of the project — Build a sensor either for Arsenic or for Coliform bacteria detection — Sense a pathogen in water — Process information with a device — Real world application à go out of the lab — Build something accessible — More than just building a biodevice! — Open-source informations — Raise awareness about water quality — Learn how to work — As a group — With people from different backgrounds — Integrate different domains of studies
- 5. Motivation The aims have been defined by our motivation as much as our motivation was dependent of the aims! — Dispose of a new way to learn — No “knowledge feeding” — Participation, initiative, try, search, solve problems… — Connect different subject around one goal — Real world aspect — Do with what we have in terms of: — Knowledge — Materials — Possibilities
- 6. Background Researches • Coliform bacteria • Arsenic • Fluorescence • Legal framework • à Decision
- 7. Coliform bacteria: General informations — Gram-negative bacteria, ferment lactose — Found in nature and in feces of warm-blooded animals — Used for fecal contamination determination — Easy to culture, especially E.Coli • Most studied coliform • Found in the intestinal tract of animals • Mostly harmless, but some strains are toxic • E.g. STEC that produces shiga-toxin, found in ruminants gut
- 8. Coliform bacteria: intoxication & detection — Symptoms of coliform intoxication: — Bloody diarrhea, vomiting — Complication: Hemolytic uremic syndrome (HUS) — HUS consists in clot formation, leading to: — blocked arteries à Ischemia — And destroyed red blood cells — E.Coli detection: — Heterotrophic plate count (HPC) is commonly used, with varying conditions (incubation, temperature, nutrient) in addition to other tests — Water considered safe under 100 cfu/ml — It has some inconvenient, so other detection techniques are being researched
- 9. Arsenic — 33rd element of the periodic table,As — AsO3, arsenic trioxyde/arsenite: most common form in the environment. — Soluble àWater can be contaminated byArsenic — Industrial origin — Geological origin
- 10. Arsenic poisoning — Interfer with Krebs cycle (inhibits pyruvate conversion to acetyl-coA) — As a slow poison, causes diseases — Skin diseases — Intestinal tract problems — Cancers — Maximum concentration advised byWHO: 10μg/L — Letal dose: 1mg/kg/day — Problem in Bangladesh and someAsian countries
- 11. Arsenic detection — Interest in detection: — Industrial devices — Academic research — Bacteria have a constitutive arsenite and arsenate detection mechanism. — Expression of a specific membrane protein complex which serves to pump the arsenite residues only when they are present. — Use of this mechanism to engineer a biosensor
- 12. Legal framework — International framework — Precaution principle — Substantial equivalence principle — Switzerland: Protection of the environment and public health — Antibiotic resistance gene à Confinement à restrictions — Sample-holder: — Transport — 3 layers — Waste gestion — It makes us aware of our responsibilities — It forces us to communicate àWe were looking what we are allowed to do and we discover that the legal demands forces us to think HOW to continue our research and build our prototype.
- 13. Fluorescence — Emission of light by a substance that has previously been excited by light at a specific wavelenght or by other electromagnetic radiation. — Green fluorescent protein: excited at 395nm, emitting green light at 509nm. — From jellyfishAequoreaVictoria — Used in biology for tracking — Expressed in the reporter bacteria after having sensedArsenic — Measure: light intensity at a specific wavelenght
- 14. Decision: the choice of fluorescence — Do with what already exists, where the most informations are available. — Work with Bangalore: students, responsive. — Use fluorescence to detectArsenic via the bioreporter — Fluorescence can also be used to detect E.Coli — Based on intrisic fluorescence of bacteria components (in the UV range) — Amino-acids — Nucleic acids Arsenic presence E.Coli senseAs Production of green fluorescence: measurable, proportional toArsenic concentration Activation of GFP gene
- 15. Design criteria General: • Portability • Low-cost • Replicability Fluorescence kit: • Light-source • Filter • Sample-holder • Receptor • Data analysis
- 16. Prototype I • Presentation • Demonstration
- 18. Our fluorometer LED Emitting at a specific wavelength Sample Detector Detecting a specific wavelength
- 19. Camera as a detector — Canon PowershotA530 — CHDK (Canon hackers development kit)
- 20. Image Processing— ImageJ, an open-source image processing software
- 21. Script — Fiji is similar to ImageJ, but allows to write scripts — Permits automation of image analysis
- 22. Tests of our device — FITC Dextran — Constitutively expressing-eGFP E.Coli — Arsenic biosensor http://apb.tbzmed.ac.ir/Portals/0/Archive/Vol2No1/Pics/2/2.Fig2.jpg
- 23. Demonstration
- 24. Improvements — Addition of a battery and a switch — To avoid using anArduino as a simple battery — The LED can be individually turned on/off — Fixation of the camera in the device — Pictures more precise — Vertical position of the sample — To allow an easiest change between different samples
- 25. Prototype II • Presentation • Demonstration
- 27. Quantification with the Photoresistance • More the light increases, more the resitance decreases so moreVout tends to equalVin • Inversely, more the light decreases, more the resistance increases so moreVout tends to be null. • Problem:The photoreistance isn’t enough sensitive.
- 28. Quantification with the light-to- frequency device 330 Ω Light to frequency device 5 V Sample holder Arduino Analogic pin 5 Thanks for using the free edition of CircuitLab! To upgrade, please visit www.CircuitLab.com/upgrade/ • The mechanisms are the same, but only the quantifier is different
- 29. Calibration • Take measures with known arsenic concentrations. • Plot them into a graph. • Light = slope * concentration + const • Concentration = (light - const) / slope
- 30. Data and analysis What we have done: Prototype 1 with dextran Prototype 1 with eGFP Prototype 2 with dextran Prototype 1 with arsenic biosensor Prototype 2 with arsenic biosensor
- 31. Prototype 1 with dextran 0 50 100 150 200 250 0 0.02 0.04 0.06 0.08 0.1 0.12 Greenlightintensity[au] Concentration of Dextran [g/L]
- 32. Prototype 1 with eGFP y = 102.3x + 3.5759 R² = 0.99917 y = 1E+07x + 415889 R² = 0.99942 1 10 100 1000 10000 100000 1000000 10000000 0.000001 0.00001 0.0001 0.001 0.01 0.1 1 GreenLightintensity[au] Sample dilution MEAN mean x area Linear (MEAN) Linear (mean x area)
- 33. Prototype 2 with dextran 0 2 4 6 8 10 12 14 16 0 10 20 30 40 50 60 Signal dextran solution [ml]
- 34. Prototype 1 with arsenic biosensor 0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 70 80 90 100 GreenLightIntensity[au] Arsenite [µg]
- 35. Prototype 2 with arsenic biosensor — Failure!
- 36. Conclusion
- 37. Future directions — Experiment more our prototypes with arsenic biosensor — Learn how the different aspects interact — Improve the devices — Test LEDs — Test filters — Add lenses — Improve reception — Improve our prototypes — Improve CHDK to do the analysis — Redesign the box to be used with a smartphone, create an app — Many samples at the same time
- 38. Future directions: General reflexions — Sample holder — Size — Environment for bacteria activity — Change the fluorescent protein — Bigger difference between excitation and emission — Longer wavelenght = cheaper LEDs — Use another reporter than GFP? — Shorten reaction time — Another arsenic measuring way? — Living matter = many parameters to manage: Bacteria number, temperature, phase, oxygen and nutrients, …
- 39. Thanks — Sachiko Hirosue — Robin Scheibler — Prof. Michaël Bensimon — Nina Buffi — José Artacho — Sabrina Leuenberger, Heinz Straessle, Charles Joye — Prof. Martial Geiser, FredericTruffer, Jean Iwanovski — Prof. Jan RoelofVan der Meer, Siham Beggah, Davide Merulla