Team:UGent Belgium/Biofunction

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Biofunction

Overview

With the biofunction group we will make several constructs to test and validate our system. We will try to enhance the function of the 3D printed shape by using biological nucleation proteins. In this way, we can improve the condensation capacity of the water collector. To achieve this, we will use the InaZ gene, an ice nucleating protein (INP) of Pseudomonas syringae. These INP’s are known to cause ice damage on plants and are also frequently used in snowmakers. Recently however, Pseudomonas syringae was also found in clouds, where they can maybe help in rain formation [1-3].

The schematic structure of INP can be found underneath. It consists of a membrane binding N-terminal domain, some internal repeating domains responsible for the nucleation, and a C-terminal domain.

INP structure

On the water collector, biotin will be coated. Hence, our constructs must contain streptavidin for effective binding this to the 3D shape. To produce and attach these INP’s to our water collector, we will investigate two options:

  1. Production of an INP-streptavidin fusion protein in E. coli, followed by lysis of the cells and extraction of the fusion-protein
  2. Separate membrane expression of both streptavidin and INP in E. coli

INP-streptavidin fusion protein

We will design a fusion protein of streptavidin with a modified INP lacking its membrane binding N-terminal domain, as depicted below.

Strep-inaZ-C

To test the function of the different parts, and the fusion protein as a whole, we will produce three control constructs:

  1. A fusion protein of GFP and streptavidin to visualize the binding of streptavidin constructs to the biotin coated shape. The GFP part is a GFP mutant that gives the brightest signal after UV excitation and works as a monomer, in contrast to normal GFP which only works as a dimer [4,5].

    strep-GFP

  2. A fusion protein of the InaZ lacking its terminal domain, GFP, and streptavidin. With this construct we can confirm that the InaZ doesn’t influence the streptavidin binding to biotin.

    strep-GFP-inaZ-C

  3. To ensure that the streptavidin is responsible for binding to the 3D printed structure, we will use a construct of the modified InaZ fused with GFP, but without streptavidin. If streptavidin is responsible for the binding to the 3D structure, the resulting protein of this construct should not be detected on the shape after applying it.

    GFP-inaZ-C

As the first two constructs both contain streptavidin, the resulting proteins from these constructs should, after lysis and extraction from E.coli, bind to the biotin on the 3D printed structure.

Membrane expression