Team:Washington/Magnetosomes/Magnet Results

From 2011.igem.org

(Difference between revisions)
(What’s in the Magnetosome Toolkit?)
(Superfolder GFP-magnetosome gene protein fusions)
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== '''Superfolder GFP-magnetosome gene protein fusions'''==
== '''Superfolder GFP-magnetosome gene protein fusions'''==
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The two genes we characterized as superfolder GFP fusions are ,<i>mamK</i> and <i>mamI</i>. They each perform core functions of magnetosome formation. MamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. MamI is a membrane-localized protein required for magnetosome vesicle formation that has also been shown to localize on the MamK filament. For more information, see the <i>mamAB</i> description [http://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions 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.''  
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The two genes we characterized as fusions with superfolder GFP are <i>mamK</i> and <i>mamI</i>. They each perform core functions of magnetosome formation. MamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. MamI is a membrane-localized protein required for magnetosome vesicle formation that has also been shown to localize on the MamK filament. For more information, see the <i>mamAB</i> description [http://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions 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===  
===mamK: Filament formation===  
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<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center>
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center>
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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.  
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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 along the long axis of the bacteria. In the our images of sfGFP-MamK, scaffold-like structures can be clearly seen running through the length of most of the cells, in some cases looping back within a single cell for "figure-8" shaped filaments. In other cases, the filaments seem to prevent the cells from dividing properly, resulting in long chains of ''E. coli''. In our experimental result, there was an over- expression of mamK which connected the ''E.coli'' cells together.  
   
   
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" >
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" >
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<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center>
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center>
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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.
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For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. This fluorescence profile is easily seen by the fluorescence profile analysis seen on the panel on the right. The graph shows that the fluorescence levels peak near the cell membrane and decrease to a minimum in the middle of the cytoplasm.
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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'' '''==

Revision as of 01:02, 29 September 2011


Magnetosome Toolkit: Results Summary


What’s in the Magnetosome Toolkit?

  • A set of 10 gene clusters from the essential mamAB operon of strain AMB-1
  • Our favorite genes as translational fusions with superfolder gfp in pGA vectors
  • A table compiling individual gene functions from our literature search




Superfolder GFP-magnetosome gene protein fusions

The two genes we characterized as fusions with superfolder GFP are mamK and mamI. They each perform core functions of magnetosome formation. MamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. MamI is a membrane-localized protein required for magnetosome vesicle formation that has also been shown to localize on the MamK filament. For more information, see the mamAB description 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 along the long axis of the bacteria. In the our images of sfGFP-MamK, scaffold-like structures can be clearly seen running through the length of most of the cells, in some cases looping back within a single cell for "figure-8" shaped filaments. In other cases, the filaments seem to prevent the cells from dividing properly, resulting in long chains of E. coli. In our experimental result, there was an over- expression of mamK which connected the E.coli cells together.


mamI: Membrane Localization

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

For mamI, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. This fluorescence profile is easily seen by the fluorescence profile analysis seen on the panel on the right. The graph shows that the fluorescence levels peak near the cell membrane and decrease to a minimum in the middle of the cytoplasm.




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 mamAB genes description page.