Team:UNIK Copenhagen/ExperimentalApproach


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<p><i>Confocal image of Magnetospirillum Gryphiswaldense</i></p>
<p>Confocal image of Magnetospirillum Gryphiswaldense</p>

Revision as of 09:07, 2 October 2013

Experimental Approach

To realize our idea of exploring and demonstrating the high biotechnological potential of the magnetosome nanoparticles, we had to find a way to execute this idea in a limited time frame and in a way that would get us results which also needed to be easily communicable to a wide audience. Our solution was the thought of linking a fluorescent protein to the magnetosome membrane. This fluorescent protein, would give us results that could be communicated with just a simple picture of our bacteria cells and could be just as easily understood by everyone. In addition it would highlight one of the big advantages magnetosomes have over other nanomagnets, they are surrounded by a lipid bilayer, a feature which we wanted to utilize. The fluorescent protein could be easily replaced by for example a drug, another protein, a dye and more to realize a wide range of possible applications in the future.

Confocal image of Magnetospirillum Gryphiswaldense

As a membrane anchor to link our fluorescent protein to the magnetosome we chose a protein called MamC that convinced us with its excellent properties. MamC is known for being highly abundant, very stable and non-interfering with the magnetosome formation itself which made it the perfect match for our experiment (Grünberg et al 2004; Scheffel et al. 2006). As our model organisms to work with in the lab, we chose two strains of magnetotactic bacteria that we saw have been also chosen by the majority of researchers working in this field. Exciting to us was that both needed different growing conditions to thrive, Magnetospirillum Gryphiswaldense needs oxygen while the other candidate Magnetospirillum magnetotacticum likes anaerobic conditions, growing them sounded like a nice challenge. For making our gene constructs of MamC plus fluorescent gene before moving them into the magnetotactic bacteria we decided to work with the well known all-rounder Escherichia coli.

We then realized that we had to choose an additional fluorescent protein to work with other than the usually utilized green fluorescent protein (GFP). GFP shows green fluorescence when exposed to blue light. Since GFP requires oxygen for its chromophore production, which is responsible for its fluorescence, it could only be used efficiently with our aerobic strain. Therefore we chose a flavin mononucleotide–based fluorescent protein (FBFP), which is able to fluoresce without needing oxygen, to use in our anaerobic strain (Drepper et al. 2007).

For fun we also picked up our own Copenhagen strain from local waters and tested out how to isolate and grow it. Further on we want to sequence its genome and compare it to the genome of other magnetotactic bacteria.

Stylized magnetotactic bacterium, where you can see the magnetosomes with the MamC protein inside its membrane

Going into the lab our strategy then was to isolate the MamC magnetosome gene from both our magnetotactic bacteria and fuse it to one of the fluorescent protein genes in a vector.

The isolated MamC and fluorescent protein genes (GFP/FBFP) are cloned together into a vector

This vector serves as a plasmid that we can transfer into our magnetotactic bacteria, which will then express our MamC protein. MamC will be fluorescently labeled so that we can make it visible under blue light in a microscope.

Here you can see the MamC protein fused with one of our fluorescent proteins, the fluorescence it emits under blue light will be visible to the bare eye

Finally when we make our bacteria express the MamC-flourescent protein construct in its magnetosomes, we can isolate them and further on study their behavior concerning future applications.

  • Drepper, Thomas, et al. "Reporter proteins for in vivo fluorescence without oxygen." Nature biotechnology 25.4 (2007): 443-445.
  • Grünberg, Karen, et al. "Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense." Applied and environmental microbiology 70.2 (2004): 1040-1050.
  • Scheffel, André, et al. "The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals." Journal of bacteriology 190.1 (2008): 377-386.