UGent Belgium PI’s: Wim Van Criekinge, Marjan De Mey, Yves...
Transcript of UGent Belgium PI’s: Wim Van Criekinge, Marjan De Mey, Yves...
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UGent_Belgium
PI’s: Wim Van Criekinge, Marjan De Mey, Yves Briers
Sofie Lodens, Michiel Stock, Sandra Steyaert, Steven De Blieck, Bob Van Hove, Bram De Jaegher, David Bouwens
Maarten Van Brempt, Bram Danneels, Griet De Clercq, Wouter Steyaert, Chari Vandenbussche
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IntroductionTackle a global problem
Water scarcity
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IntroductionWhere to get more fresh water?
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StrategyHow to harvest atmospheric water?
Create 3D-printed optimized shape Coat with biological nucleation proteinsfor water condensation and collection to improve water condensation
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Strategy3D-printed optimized shape for water condensation and collection
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StrategyHow to harvest atmospheric water?
Create 3D-printed optimized shape Coat with biological nucleation proteinsfor water condensation and collection to improve water condensation
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StrategyHow to coat a protein on a printed structure?
- PLA + biotine
- Fusion protein with streptavidin
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StrategyHow to harvest atmospheric water?
Create 3D-printed optimized shape Coat with biological nucleation proteinsfor water condensation and collection to improve water condensation
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StrategyCondensation promoting protein?
… meet the Pseudomonas syringae Ice Nucleating Protein (INP)
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StrategyCondensation promoting protein?
… meet the Pseudomonas syringae Ice Nucleating Protein (INP)
Recently Pseudomonas syringae was alsofound in clouds, where it might help theformation of rain by acting as a condenationnucleus
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Work packages
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WP1. Shape
WP2. Filament
WP3. BioFunction
WP4. Functional Assay
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WP 1: Shape
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WP 2: Filament
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Biotinylated PLA
a) Chemical synthesis → too complicated
b) Impregnated and remolten PLA:
• Dissolve PLA + biotin in hot DMF
• Crash out PLA by adding to EtOH
• Wash with biotin saturated EtOH
• Extrude new filament
c) Coat with ‘biotin paint’:
• DCM saturated with PLA, suspended biotin
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WP 2: Filament
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Test for addition of biotin: HABA/avidin assay
Coated Impregnated Untreated
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WP 3: BioFunction
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Whole-cell Protein extract
Control
Functional
Setup constructs
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WP 3: BioFunction
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The making of …
A monomeric GFPuv (mGFPuv) A GG-safe Ice Nucleating Protein
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WP 3: BioFunction
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The making of …
And a truncated Ice Nucleating Protein(INP-RC fusion proteins with GFP/streptavidin)
“Analogous ice forming movie”
1) inaZ(RC)-mSA2 3) mGFPuv-Strep 5) mGFPuv2) inaZ(RC)-mGFPuv 4) mGFPuv-mSA2
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WP 4: Functional AssayDoes it stick?
PLA filament+biotine PLA+biotine coated glass plate
Left: mGFPuv – mStrep ?10: mGFPuv – mStrep Right: a control..11: mGFPuv
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Whole-cell approach no visible fluorescence(data not shown)
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WP 4: Functional AssayDoes it stick?
… a more sensitive assay
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Histag
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WP 4: Functional AssayDoes it make water?
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