Team:Washington/Magnetosomes/Magnet Results

From 2011.igem.org

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=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==
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After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKL, mamMNOPA, and mamQRBSTUV. The PCR products of the first, and the third part of the assembly are shown below. Both fragments of the assembly have been partially sequenced confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when this samples were imaged, filaments in the first part (mamHIEJKL)were still apparent. <br/>
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After verifying that the construction of the sfGFP-MamK scaffold worked as expected, we proceeded to create a full assembly of the <i>mamAB</i> operon by building three super-assemblies: '''mamHIEJKL''', '''mamMNOPA''', and '''mamQRBSTUV'''. The PCR products of these intermediate assemblies are shown below. The '''mamHIEJKL''' and '''mamQRBSTUV''' have been partially sequence-confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when cells with the '''mamHIEJKL''' construct were imaged, they appeared to be forming chains.<br/>
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|500px|middle]]:[[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|500px|middle]]:[[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]
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Revision as of 00:37, 29 September 2011


Magnetosome Toolkit: Results Summary


What’s in the Magnetosome Toolkit?

  • Our favorite genes in pGA vectors
  • A set of the 18 essential genes for the various steps of magnetosome formation
  • A table compiling individual gene functions from our literature search



Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions

As previously stated, 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 loss of mamK inhibits membrane formation. (for other gene functions, please see the iGEM Toolkits parts submitted page)Using our two genes of interest, we created C-terminal sfGFP fusions so we could track the localization of each gene separately within E.coli.

mamK: Filament formation


Washington igem11 MamK fusion full 01.jpgWashington igem11 MamK fusion gfp 01.jpg

The results we obtained with our sfGFP fusions inside E.coli were comparable to those done through other studies in the host organism Magnetospirillum magneticum. Within AMB-1, mamK is a filament which runs through the length of the bacteria. In the our images of mamK, filamentous structures can be clearly seen running through the length of many bacteria. In our experimental result, there was an over- expression of mamK which connected the E.coli cells together.

More images of E.coli with sfGFP mamK fusion
Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg
Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg
Washington SfGFP K 4C5-col1 03 crop.jpg
Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg
Washington igem11 SfGFP K 4C5-col1 05 crop.jpg
Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg
Washington igem11 SfGFP K 4C5-col1 04 crop.jpg
Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg


mamI: Membrane Localization

Washington igem11 MamIfusion full.jpgWashington igem11 MamIfusion GFP.jpgWashington igem11 mamI graph.png

For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This can be seen in the fluorescence profile analysis that was taken while imaging the cells. The graph shows that as the arrow crosses the cell membrane, the fluorescent peaks are at a maximum, and through the center of the cell, the level of fluorescence decreases.



Construction of the R5 region of the Magnetosome Island in E.coli

After verifying that the construction of the sfGFP-MamK scaffold worked as expected, we proceeded to create a full assembly of the mamAB operon by building three super-assemblies: mamHIEJKL, mamMNOPA, and mamQRBSTUV. The PCR products of these intermediate assemblies are shown below. The mamHIEJKL and mamQRBSTUV have been partially sequence-confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when cells with the mamHIEJKL construct were imaged, they appeared to be forming chains.

Washington iGEM2011 magentosome HIEJKL3k3.png:Washington iGEM2011 magentosome MNOPA.pngWashington iGEM2011 magentosome QRBSTUV.png

A set of the 18 essential genes for the various steps of magnetosome formation

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 groups:

Gel Extracts of Individual Magnetosome Genes
Gene groups Length (bp)
mamHI 1541
mamE 2172
mamJ 1538
mamKL 1336
mamMN 2323
mamO 1914
mamPA 1493
mamQRB 2029
mamSTU 2030
mamV 1002
.


A table of individual gene functions

Please see our mam gene descriptions page.

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