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

As cell lysis and protein extraction is laborious and resource intensive we will also investigate the possibility to express both streptavidin and INP on the outer membrane surface of E. coli. For this purpose, two constructs will be made:

  1. A custom made fusion protein of streptavidin with the membrane binding region of INP

    N-C-inaZ-strep

    As an alternative we could use an existing biobrick of streptavidin fused to the membrane binding domain Lpp-OmpA

    Lpp-OmpA-strep

  2. The wild type (WT) InaZ

    INP structure WT

In this approach, E. coli will bind to the biotin on the surface of the 3D printed structure using the membrane-bound streptavidin. The WT InaZ (containing the membrane binding domain) can then perform its normal nucleation function.

Again, several control constructs will be used to test the function of the different parts:

  1. A custom made fusion protein of GFP with the membrane binding region of INP. This will allow us to visually assess whether the membrane binding region of InaZ functions properly as display.

    N-C-inaZ-GFP

    Alternatively, we will use a fusion of Lpp-OmpA to check its functionality as display.

    Lpp-Ompa-GFP

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

    N-C-inaZ-GFP-strep

  3. Similar to the previous construct, but with Lpp-OmpA

    Lpp-OmpA-GFP-strep

To make the constructs for these fusion proteins, we can use some existing biobricks to start from:

InaZ membrane binding region:
BBa_K811003
Lpp-OmpA - Streptavidin fusion protein:
BBa_J36846
WT InaZ:
BBa_K584024

Other necessary building blocks can be ordered as gblocks, and can be easily cloned to construct the necessary fusion proteins. Restriction sites will be included to ensure compatibility with golden gate, biobricks and bglbricks.

References

  1. DeLeon-Rodriguez, N. et al. Microbiome of the upper troposphere: Species composition and prevalence, effects of tropical storms, and atmospheric implications. Proc. Natl. Acad. Sci. 110, 2575–2580 (2013).
  2. Pratt, K. A. et al. In situ detection of biological particles in cloud ice-crystals. Nat. Geosci. 2, 398–401 (2009).
  3. Lundheim, R. Physiological and ecological significance of biological ice nucleators. Philos. Trans. R. Soc. B Biol. Sci. 357, 937–943 (2002).
  4. von Stetten, D., Noirclerc-Savoye, M., Goedhart, J., Gadella, T. W. J. & Royant, A. Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K. Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 68, 878–82 (2012).
  5. Ito, Y., Suzuki, M. & Husimi, Y. A novel mutant of green fluorescent protein with enhanced sensitivity for microanalysis at 488 nm excitation. Biochem. Biophys. Res. Commun. 264, 556–60 (1999).