Team:Washington/Magnetosomes/Magnet Toolkit

From 2011.igem.org

Magnetosome Toolkit

What are magnetosomes? Where do they come from?

A Chain of Magnetosomes within Magnetospirillum magneticum AMB-1

Magnetotactic Bacteria are prokaryotic organisms which possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome formation. Magnetosomes are small invaginations of the bacterial cell membrane that contain magnetite particles

These particles range in size between 20 and several hundred nanometers and are aligned in one or several chains along the long axis of the bacteria. These particles act together to form a magnetic dipole across the bacteria, allowing it to perceive the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, the magnetosome is currently thought to help aid the organism in its search for the perfect oxygen level from a three dimensional space (in all directions) to a one dimensional space along a single path.

A Closer look at Magnetosome Formation

The formation of the magnetosome organelle is a highly regulated, step-wise process requiring a cascade of essential genes. The process is generally hypothesized as four stages: i) membrane invagination, ii) acquiring minerals for magnetite formation, iii) iron-oxidation and reduction, iv) magnetite nucleation and morphology regulation. Earlier gene products must be present for later gene products to be formed as shown in the diagram below: [http://www.pnas.org/content/107/12/5593.full.pdf+html]:

Diagram of stepwise magnetosome construction within AMB-1.

What did the UW iGEM team do with Magnetotactic Bacteria?

It is thought that many of the essential genes associated with magnetosome formation are located within a well-conserved region known as the magnetosome island (MAI). The MAI consists of 14 gene clusters labeled R1-R14 (see diagram below).Our team focused on the genes of the mamAB gene cluster (R5) as they were previously shown to be essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].

The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in E.coli. This was done in order to understand more about magnetosome formation and the magnet synthesis mechanism because many of the genes' functions are still unknown in the host species. Using the information we have gained, we have organized a Magnetosome Toolkit containing most of the essential genes for proper magnetosome formation. Ultimately, we would like to continue expanding the magnetosome toolkit to have enough parts to show complete magnetosome formation within E.coli.

The mamAB operon (R5) located in the magnetosome island (MAI).

About the Magnetosome Toolkit:

Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team was able to create a "Magnetosome Toolkit" consisting of the most basic parts required for magnetosome formation. Providing this toolkit will help allow future iGem teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in various types of bacteria.

Toolkit construction and mamAB assembly in E.coli

What's in the toolkit

Before piecing together the 16 kb genome of the mamAB gene cluster within the magnetosome island (MAI), we extracted out the genes in the following group:

Using standard PCR protocols, these genes were extracted and..... These genes were visualized on gels and sequence confirmed; therefore, we can be sure that these are some of the many genes required for proper magnetosome formation.

As previously noted, magnetosome formation within the host-organism, Magnetospirillium magneticum, strain AMB-1, is a highly regulated step-wise process. As shown in (link: figure #), genes encode for an invagination in the inner membrane, there are genes which help align the magnetosomes into their characteristics chains, and there are genes which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment, since it has many practical uses in synthetic biology (??? maybe we could make a link to the alkenes future if they wanted to use mamI....)

Our genes of interest were mamK and mamI as they have functions related to localization of the magnetosome. Specifically, mamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. mamK is also shown to localize the mamI, which is loss inhibits membrane formation. For more information, refer to the table below:

Using our two genes of interest, we wanted to create C-terminal sfGFP fusions so we could track the localization of each gene within E.coli.

To begin, we preformed a Gibson reaction using our genes from the Magnetosome toolkit, sfGFP, and a 1C3 vector with a pLac promoter. After the standard protocols of Gibson, transformation and plating, we imaged the samples and were able to see that the gene localizations in the strain AMB-1 were also shown in E.coli.