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

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

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

WP 2: Filament

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Test for addition of biotin: HABA/avidin assay

Coated Impregnated Untreated

WP 3: BioFunction

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Whole-cell Protein extract

Control

Functional

Setup constructs

WP 3: BioFunction

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The making of …

A monomeric GFPuv (mGFPuv) A GG-safe Ice Nucleating Protein

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

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)

WP 4: Functional AssayDoes it stick?

… a more sensitive assay

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Histag

WP 4: Functional AssayDoes it make water?

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