Difference between revisions of "Team:Munich/Results"

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<h2>
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Optimizing a Cell-Free System
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</h2>
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<h3>
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Cultivation
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</h3>
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<h3>
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Lysis
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</h3>
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<h3>
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Processing
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</h3>
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<h3>
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Quality Control
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</h3>
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<h2>
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Bacteriophage Assembly
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</h2>
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<h3>
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Home-Made Systems Can Be Compared To Commercial Systems
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</h3>
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<h3>
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Cell-Free Systems Allow Host Independent Bacteriophage Assembly
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</h3>
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<h3>
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Quality Control Of Bacteriophages
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</h3>
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<h3>
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Assembly Of Clinically Relevant Bacteriophages
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</h3>
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<h3>
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Sequ-Into Complements Continuous Engineering Cycles in Phactory
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</h3>
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<h2>
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Modular Bacteriophage Composition
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</h2>
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<h2>
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Isolation
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</h2>
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<h3>
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Phenol-Chloroform Precipitation Achieves High Titers
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</h3>
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<h2>
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Encapsulation
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</h2>
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<h3>
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Bacteriophages Encapsulated In Alginate Can Withstand Gastric Acid
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</h3>
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<p>
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Phactory yields phages with toxicity levels that allow oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice, while direct intravenous application requires additional purification steps. To overcome these hurdles, we prototyped two 3D-printed fluidic devices that can be assembled for less than $5. For oral application, we built a nozzle to encapsulate the phages in monodisperse calcium-alginate microspheres protecting them in the stomach. The alginate solution, ejected from a dispenser needle with a syringe pump, was sheared off by a parallel stream of air. Our results show that after 1 hour incubation in simulated gastric fluid, active phages are successfully released in simulated intestinal fluid. For intravenous administration, we can purify the bacteriophages from the remaining cell-extract via fractionation in a pressure-driven size-exclusion filter system. Additionally, we built microfluidic hardware for our human practice project OraColi. </p>
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<p> - Alginat für lokale pH wert erhöhung im magen und auflösung in chelatoren(darm), da Ca2+ Ionen quervernetzen</p>
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<p> - Enkapsulierung durch Co-Flow System. Druckluft schert Tropfen von Spritzennadel ab, Düse == Hardware  Quervernetzung 1h in CaCl2 Lsg  Waschen  Verifikation durch Mikroskopie  Fertig für Einsatz (insg. Ca 1.5-2h)</p>
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<p>
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Experimente
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1. 3D Modell von SYBR Gold gelabelten Phagen im Droplet durch z-Stack (BF/GFP)
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2. Darkfield Bilder von gelabelten Droplets (bild)
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3. Droplet Zusammensetzung (Viskosität zweier Alginate)
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4. Droplet Größen bei Flussrate und Druck
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5. Verhalten im Magen bzw. saurem Millieu (pH: 1) und Pepsin (1h @37°C, 10h @ RT) -> Phagen Degradation (barplot)
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6. Verhalten im Darm bzw. pH 7 und Pankreatin (2h @ 37°C) ->Phagen relase über Zeit (plot)
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</p>
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<div id="phareferences" class="row">
 
<div id="phareferences" class="row">

Revision as of 01:25, 16 October 2018

Phactory

Results

First Blick of Phactory


A generic square placeholder image with rounded corners in a figure.
BIG BROTHER IS WATCHING YOU

Optimizing a Cell-Free System

Cultivation

Lysis

Processing

Quality Control

Bacteriophage Assembly

Home-Made Systems Can Be Compared To Commercial Systems

Cell-Free Systems Allow Host Independent Bacteriophage Assembly

Quality Control Of Bacteriophages

Assembly Of Clinically Relevant Bacteriophages

Sequ-Into Complements Continuous Engineering Cycles in Phactory

Modular Bacteriophage Composition

Isolation

Phenol-Chloroform Precipitation Achieves High Titers

Encapsulation

Bacteriophages Encapsulated In Alginate Can Withstand Gastric Acid

Phactory yields phages with toxicity levels that allow oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice, while direct intravenous application requires additional purification steps. To overcome these hurdles, we prototyped two 3D-printed fluidic devices that can be assembled for less than $5. For oral application, we built a nozzle to encapsulate the phages in monodisperse calcium-alginate microspheres protecting them in the stomach. The alginate solution, ejected from a dispenser needle with a syringe pump, was sheared off by a parallel stream of air. Our results show that after 1 hour incubation in simulated gastric fluid, active phages are successfully released in simulated intestinal fluid. For intravenous administration, we can purify the bacteriophages from the remaining cell-extract via fractionation in a pressure-driven size-exclusion filter system. Additionally, we built microfluidic hardware for our human practice project OraColi.

- Alginat für lokale pH wert erhöhung im magen und auflösung in chelatoren(darm), da Ca2+ Ionen quervernetzen

- Enkapsulierung durch Co-Flow System. Druckluft schert Tropfen von Spritzennadel ab, Düse == Hardware  Quervernetzung 1h in CaCl2 Lsg  Waschen  Verifikation durch Mikroskopie  Fertig für Einsatz (insg. Ca 1.5-2h)

Experimente 1. 3D Modell von SYBR Gold gelabelten Phagen im Droplet durch z-Stack (BF/GFP) 2. Darkfield Bilder von gelabelten Droplets (bild) 3. Droplet Zusammensetzung (Viskosität zweier Alginate) 4. Droplet Größen bei Flussrate und Druck 5. Verhalten im Magen bzw. saurem Millieu (pH: 1) und Pepsin (1h @37°C, 10h @ RT) -> Phagen Degradation (barplot) 6. Verhalten im Darm bzw. pH 7 und Pankreatin (2h @ 37°C) ->Phagen relase über Zeit (plot)

References

  1. Abudayyeh, O.O. et al., 2016. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573.
  2. Boothby, T.C., Tapia, H., Brozena, A.H., Piszkiewicz, S., Smith, A.E., Giovannini, I., Rebecchi, L., Pielak, G.J., Koshland, D., and Goldstein, B., 2017. Tardigrades use intrinsically disordered proteins to survive desiccation. Mol Cell 65, 975ñ984.
  3. Centers for Disease Control and Prevention, 2017. Antibiotic/Antimicrobial Resistance.
  4. GDDiergezondheid, 2017. Mastitis (uierontsteking).
  5. Gootenberg, J.S. et al., 2017. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438ñ442.
  6. Jia, B. et al., 2017. CARD 2017: Expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Research 45(D1), D566ñD573.
  7. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E., 2012. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816-821.
  8. Liu, L. et al., 2017. The molecular architecture for RNA-guided RNA cleavage by Cas13a. Cell 170, 714ñ726.e10.
  9. McArthur, A.G. et al., 2013. The comprehensive antibiotic resistance database. Antimicrobial Agents and Chemotherapy 57, 3348ñ3357.
  10. McArthur, A.G. and Wright, G.D., 2015. Bioinformatics of antimicrobial resistance in the age of molecular epidemiology. Current Opinion in Microbiology 27, 45ñ50.
  11. Schwechheimer, C., and Kuehn, M.J., 2015. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews: Microbiology 13, 605-619.
  12. Sloan, D., Batista, A., and Loeb, A., 2017. The Resilience of Life to Astrophysical Events. Scientific Reports 7, 5419-5424.
  13. Statistics Netherlands, 2015. Dutch dairy in figures.
  14. World Health Organization, 2016. Antibiotic resistance.
  15. Zetsche, B, Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., van der Oost, J., Regev, Aviv, Koonin, E.V., Zhang, F., 2015. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163, 759-771.