Team:Washington/Magnetosomes/Results

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Revision as of 19:45, 19 September 2011

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 allows future iGem teams to manipulate and understand magnetosome formation to one day create magnets in various types of bacteria.

What' in the toolkit and How We Assembled It

After 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:

Gene groups	Length (bp)
mamHI	1541
mamE	2172
mamJ	1538
mamKL	1336
mamMN	2323
mamO	1914
mamPA	1493
mamQRB	2029
mamSTU	2030
mamV	1002

.

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:

Gene	AMB Number	Cluster Membership	Member of 28 genes list? (specific/related*)	Function Summary (Vesicle chain formation, and/or biomineralization)	Gene Function
mamH	amb0961	mamAB	Related
mamI	amb0962	mamAB	Specific	Vesicle, (Chain Formation?)	>berkeley 2010: Loss causes no membrane formation, is localized onto chains
mamE	amb0963	mamAB; mam Islet	Related		>Membrane-bound serine protease required for magnetite formation; might control the localization of other magnetosome proteins
mamJ	amb0964	mamAB; mam Islet	Specific	Chain Formation	>Proper magnetosome chain organization/assembly
mamK	amb0965	mamAB; mam Islet	Related	Chain Formation	>required for proper magnetosome chain organization; *bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain, shown to localize the mamI
mamL	amb0966	mamAB; mam Islet	Specific	Vesicle, biomineralization	>berkely 2010: Crucial to mangneosome membrane creation, shown to be spread across the cell membrane and sometimes forms lines
mamM	amb0967	mamAB	Related		>biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome
mamN	amb0968	mamAB	Related		>biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome
mamO	amb0969	mamAB	Related		>biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome
mamP	amb0970	mamAB	Related	Biomineralization	>berkeley 2010: loss causes weak magnetic response, with large but fewer crystals
mamA	amb0971	mamAB	Related		>Required for magnetosome activation; activation of vessicles
mamQ	amb0972	mamAB; mam Islet	Related		>ORF; formation/maintenance of magnetosome membranes
mamR	amb0973	mamAB	Specific	Chain formation, Biomineralization	>ORF; plays a role in controlling both particle number and size of magnetite cyrstals
mamB	amb0974	mamAB	Related	Vesicle, Biomineralization	>indirect role in magnetosome membrane invagination and biomineralization; magnetosome compartment formation
mamS	amb0975	mamAB	Specific
mamT	amb0976	mamAB	Specific	Biomineralization	>magnetite crystal growth; participates in different steps during magnetite synthesis
mamU	amb0977	mamAB	Related
mamV	amb0978	mamAB	N/A

.

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.

sfGFP fusions of mamK and MamI in both AMB-1 and *E.coli*.
	Strain AMB-1	E.coli
mamK-sfGFP	Cell 2	Cell 3
mamI-sfGFP	Cell B	Cell C

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 ===About the Magnetosome Toolkit:===
-[[File:Washington Methode image.jpg|center|955px]]
 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 allows future iGem teams to manipulate and understand magnetosome formation to one day create magnets in various types of bacteria.