http://2011.igem.org/wiki/index.php?title=Special:Contributions/Saushun&feed=atom&limit=50&target=Saushun&year=&month=2011.igem.org - User contributions [en]2024-03-28T19:00:43ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-29T00:26:11Z<p>Saushun: /* A table of Translational Efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of Translational Efficiency using RBS calculator<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
== ''' A table of Translational Efficiency''' ==<br />
Using the RBS Calculator developed by the Salis, Mirsky, and Voigt ([https://salis.psu.edu/software/doReverseRBS Link]), we were able to estimate the relative translation rate of each gene from the mamAB operon.<br />
<br />
{| class="wikitable" |left<br />
|-<br />
! Gene <br />
! AMB-1<br />
! E.coli<br />
! Change in %<br />
! Actual sequence inputted in the calculator (first 9nt and last 9nt)<br />
|-<br />
| mamH<br />
| 10821.63<br />
|10821.63<br />
| 0%<br />
| TCGGAGGTG......CCAGCACAA<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
| 0%<br />
| TCGCTCTGC.....ATTGCTGGG<br />
|-<br />
| mamE<br />
| 37.82<br />
| 37.82<br />
| 0%<br />
| ATGGCCATG.....CTATCTGAT<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
| 0%<br />
| ACCGCAATG.....CCAGGGTGA<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
| 0%<br />
| GTCATTTAG.....TGGCATCGA<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
| 0%<br />
| CGGGGATAC.....CGTGCTGTT<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
| 0%<br />
| GTCGGGGCT.....CGGCCTGGC<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
| -44.29%<br />
| GAAGTCATG.....CGCCGTTAT<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
| 0%<br />
| TCCGAATGA.....CGTGTTTTG<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
| 0%<br />
| TGATGATCA.....TGTTCTGGC<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
| 0%<br />
| TAAAGTGAT.....CGAGGTCAC<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
| -44.27%<br />
| CCTTGCCGC.....TCCTTCATA<br />
|-<br />
| mamR<br />
| 8.04<br />
| 8.04<br />
| 0%<br />
| TCTCAACCA.....CGTGCAGGG<br />
|-<br />
| mamB<br />
| 16.56<br />
| 16.56<br />
| 0%<br />
| TCCAATCTT.....TGGGCCTTT<br />
|-<br />
| mamS<br />
| 32.86<br />
| 25.09<br />
| -23.65%<br />
| CACGGGCCT.....GATGGCCGA<br />
|-<br />
| mamT<br />
| 81.16<br />
| 81.16<br />
| 0%<br />
| TGGTGCAGT.....CTTGGGGAT<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
| 0%<br />
| CGCTGACCA.....CGCCTGCCT<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
| 0%<br />
| AATAAACCA.....TCGTGACCG<br />
|}.<br />
<br />
We tested this against the anti-Shine-Dalgarno 16S rRNA regions from ''E. coli'' and AMB-1, which differ only by one base – ACCTCCTTA and ACCTCCTTT, respectively. Furthermore, by modifying the native sequences, the translation initiation rates could be changed to get the proper expression levels.<br />
<br />
Each number in the table represents the translation initiation rate (au) on a proportional scale from 0.1 to 100,00+. The higher the number the more proteins are translated per mRNA. It is important to keep in mind that this RBS calculator gives an estimation of translational efficiency only.<br />
<br />
Due to the similarity in 16S rRNA sequences between the two strains, we expected to see a similar translation rates in ''E. coli'' compared with AMB-1 and the predictions fall within those expectations. However, mamN, mamQ, and mamS may have significantly lower translation rates in ''E. coli'' than AMB-1. <br />
<br />
With the use the RBS calculator, the activity of individual genes’ can be estimated thus the level gene expression can be varied by sequence modification.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-29T00:25:34Z<p>Saushun: /* A table of Translational Efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of Translational Efficiency using RBS calculator<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
== ''' A table of Translational Efficiency''' ==<br />
Using the RBS Calculator developed by the Salis, Mirsky, and Voigt ([https://salis.psu.edu/software/doReverseRBS Link]), we were able to estimate the relative translation rate of each gene from the mamAB operon.<br />
<br />
{| class="wikitable" |left<br />
|-<br />
! Gene <br />
! AMB-1<br />
! E.coli<br />
! Change in %<br />
! Actual sequence inputted in the calculator (first 9nt and last 9nt)<br />
|-<br />
| mamH<br />
| 10821.63<br />
|10821.63<br />
| 0%<br />
| TCGGAGGTG......CCAGCACAA<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
| 0%<br />
| TCGCTCTGC.....ATTGCTGGG<br />
|-<br />
| mamE<br />
| 37.82<br />
| 37.82<br />
| 0%<br />
| ATGGCCATG.....CTATCTGAT<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
| 0%<br />
| ACCGCAATG.....CCAGGGTGA<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
| 0%<br />
| GTCATTTAG.....TGGCATCGA<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
| 0%<br />
| CGGGGATAC.....CGTGCTGTT<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
| 0%<br />
| GTCGGGGCT.....CGGCCTGGC<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
| -44.29%<br />
| GAAGTCATG.....CGCCGTTAT<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
| 0%<br />
| TCCGAATGA.....CGTGTTTTG<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
| 0%<br />
| TGATGATCA.....TGTTCTGGC<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
| 0%<br />
| TAAAGTGAT.....CGAGGTCAC<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
| -44.27%<br />
| CCTTGCCGC.....TCCTTCATA<br />
|-<br />
| mamR<br />
| 8.04<br />
| 8.04<br />
| 0%<br />
| TCTCAACCA.....CGTGCAGGG<br />
|-<br />
| mamB<br />
| 16.56<br />
| 16.56<br />
| 0%<br />
| TCCAATCTT.....TGGGCCTTT<br />
|-<br />
| mamS<br />
| 32.86<br />
| 25.09<br />
| -23.65%<br />
| CACGGGCCT.....GATGGCCGA<br />
|-<br />
| mamT<br />
| 81.16<br />
| 81.16<br />
| 0%<br />
| TGGTGCAGT.....CTTGGGGAT<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
| 0%<br />
| CGCTGACCA.....CGCCTGCCT<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
| 0%<br />
| AATAAACCA.....TCGTGACCG<br />
|}.<br />
<br />
We tested this against the anti-Shine-Dalgarno 16S rRNA regions from ''E. coli'' and AMB-1, which differ only by one base – ACCTCCTTA and ACCTCCTTT, respectively. Furthermore, by modifying the native sequences, the translation initiation rates could be changed to get the proper expression levels.<br />
<br />
Each number in the table represents the translation initiation rate (au) on a proportional scale from 0.1 to 100,00+. The higher the number the more proteins are translated per mRNA. It is important to keep in mind that this RBS calculator gives an estimation of translational efficiency only.<br />
<br />
Due to the similarity in 16S rRNA sequences between the two strains, we expected to see a similar translation rates in E. coli compared with AMB-1 and the predictions fall within those expectations. However, mamN, mamQ, and mamS may have significantly lower translation rates in ''E. coli'' than AMB-1. <br />
<br />
With the use the RBS calculator, the activity of individual genes’ can be estimated thus the level gene expression can be varied by sequence modification.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-29T00:22:56Z<p>Saushun: /* A Table of Translational Efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of Translational Efficiency using RBS calculator<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
== ''' A table of Translational Efficiency''' ==<br />
Using the RBS Calculator developed by the Salis, Mirsky, and Voigt ([https://salis.psu.edu/software/doReverseRBS Link]), we were able to estimate the relative translation rate of each gene from the mamAB operon.<br />
<br />
{| class="wikitable" |left<br />
|-<br />
! Gene <br />
! AMB-1<br />
! E.coli<br />
! Change in %<br />
! Actual sequence inputted in the calculator (first 9nt and last 9nt)<br />
|-<br />
| mamH<br />
| 10821.63<br />
|10821.63<br />
| 0%<br />
| TCGGAGGTG......CCAGCACAA<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
| 0%<br />
| TCGCTCTGC.....ATTGCTGGG<br />
|-<br />
| mamE<br />
| 37.82<br />
| 37.82<br />
| 0%<br />
| ATGGCCATG.....CTATCTGAT<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
| 0%<br />
| ACCGCAATG.....CCAGGGTGA<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
| 0%<br />
| GTCATTTAG.....TGGCATCGA<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
| 0%<br />
| CGGGGATAC.....CGTGCTGTT<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
| 0%<br />
| GTCGGGGCT.....CGGCCTGGC<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
| -44.29%<br />
| GAAGTCATG.....CGCCGTTAT<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
| 0%<br />
| TCCGAATGA.....CGTGTTTTG<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
| 0%<br />
| TGATGATCA.....TGTTCTGGC<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
| 0%<br />
| TAAAGTGAT.....CGAGGTCAC<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
| -44.27%<br />
| CCTTGCCGC.....TCCTTCATA<br />
|-<br />
| mamR<br />
| 8.04<br />
| 8.04<br />
| 0%<br />
| TCTCAACCA.....CGTGCAGGG<br />
|-<br />
| mamB<br />
| 16.56<br />
| 16.56<br />
| 0%<br />
| TCCAATCTT.....TGGGCCTTT<br />
|-<br />
| mamS<br />
| 32.86<br />
| 25.09<br />
| -23.65%<br />
| CACGGGCCT.....GATGGCCGA<br />
|-<br />
| mamT<br />
| 81.16<br />
| 81.16<br />
| 0%<br />
| TGGTGCAGT.....CTTGGGGAT<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
| 0%<br />
| CGCTGACCA.....CGCCTGCCT<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
| 0%<br />
| AATAAACCA.....TCGTGACCG<br />
|}.<br />
<br />
We tested this against the anti-Shine-Dalgarno 16S rRNA regions from E. coli and AMB-1, which differ only by one base – ACCTCCTTA and ACCTCCTTT, respectively. Furthermore, by modifying the native sequences, the translation initiation rates could be changed to get the proper expression levels.<br />
<br />
Each number in the table represents the translation initiation rate (au) on a proportional scale from 0.1 to 100,00+. The higher the number the more proteins are translated per mRNA. It is important to keep in mind that this RBS calculator gives an estimation of translational efficiency only.<br />
<br />
Due to the similarity in 16S rRNA sequences between the two strains, we expected to see a similar translation rates in E. coli compared with AMB-1 and the predictions fall within those expectations. However, mamN, mamQ, and mamS may have significantly lower translation rates in E. coli than AMB-1. <br />
<br />
With the use the RBS calculator, the activity of individual genes’ can be estimated thus the level gene expression can be varied by sequence modification.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-29T00:22:15Z<p>Saushun: /* A Table of Translational Efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of Translational Efficiency using RBS calculator<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
== ''' A Table of Translational Efficiency''' ==<br />
Using the RBS Calculator developed by the Salis, Mirsky, and Voigt ([https://salis.psu.edu/software/doReverseRBS Link]), we were able to estimate the relative translation rate of each gene from the mamAB operon.<br />
<br />
{| class="wikitable" |left<br />
|-<br />
! Gene <br />
! AMB-1<br />
! E.coli<br />
! Change in %<br />
! Actual sequence inputted in the calculator (first 9nt and last 9nt)<br />
|-<br />
| mamH<br />
| 10821.63<br />
|10821.63<br />
| 0%<br />
| TCGGAGGTG......CCAGCACAA<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
| 0%<br />
| TCGCTCTGC.....ATTGCTGGG<br />
|-<br />
| mamE<br />
| 37.82<br />
| 37.82<br />
| 0%<br />
| ATGGCCATG.....CTATCTGAT<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
| 0%<br />
| ACCGCAATG.....CCAGGGTGA<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
| 0%<br />
| GTCATTTAG.....TGGCATCGA<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
| 0%<br />
| CGGGGATAC.....CGTGCTGTT<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
| 0%<br />
| GTCGGGGCT.....CGGCCTGGC<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
| -44.29%<br />
| GAAGTCATG.....CGCCGTTAT<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
| 0%<br />
| TCCGAATGA.....CGTGTTTTG<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
| 0%<br />
| TGATGATCA.....TGTTCTGGC<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
| 0%<br />
| TAAAGTGAT.....CGAGGTCAC<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
| -44.27%<br />
| CCTTGCCGC.....TCCTTCATA<br />
|-<br />
| mamR<br />
| 8.04<br />
| 8.04<br />
| 0%<br />
| TCTCAACCA.....CGTGCAGGG<br />
|-<br />
| mamB<br />
| 16.56<br />
| 16.56<br />
| 0%<br />
| TCCAATCTT.....TGGGCCTTT<br />
|-<br />
| mamS<br />
| 32.86<br />
| 25.09<br />
| -23.65%<br />
| CACGGGCCT.....GATGGCCGA<br />
|-<br />
| mamT<br />
| 81.16<br />
| 81.16<br />
| 0%<br />
| TGGTGCAGT.....CTTGGGGAT<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
| 0%<br />
| CGCTGACCA.....CGCCTGCCT<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
| 0%<br />
| AATAAACCA.....TCGTGACCG<br />
|}.<br />
<br />
We tested this against the anti-Shine-Dalgarno 16S rRNA regions from E. coli and AMB-1, which differ only by one base – ACCTCCTTA and ACCTCCTTT, respectively. Furthermore, by modifying the native sequences, the translation initiation rates could be changed to get the proper expression levels.<br />
<br />
Each number in the table represents the translation initiation rate (au) on a proportional scale from 0.1 to 100,00+. The higher the number the more proteins are translated per mRNA. It is important to keep in mind that this RBS calculator gives an estimation of translational efficiency only.<br />
<br />
Due to the similarity in 16S rRNA sequences between the two strains, we expected to see a similar translation rates in E. coli compared with AMB-1 and the predictions fall within those expectations. However, mamN, mamQ, and mamS may have significantly lower translation rates in E. coli than AMB-1. <br />
<br />
With the use the RBS calculator, the activity of individual genes’ can be estimated thus the level gene expression can be varied by sequence modification.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-29T00:20:33Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of Translational Efficiency using RBS calculator<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
== ''' A Table of Translational Efficiency''' ==<br />
<br />
{| class="wikitable" |left<br />
|-<br />
! Gene <br />
! AMB-1<br />
! E.coli<br />
! Change in %<br />
! Actual sequence inputted in the calculator (first 9nt and last 9nt)<br />
|-<br />
| mamH<br />
| 10821.63<br />
|10821.63<br />
| 0%<br />
| TCGGAGGTG......CCAGCACAA<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
| 0%<br />
| TCGCTCTGC.....ATTGCTGGG<br />
|-<br />
| mamE<br />
| 37.82<br />
| 37.82<br />
| 0%<br />
| ATGGCCATG.....CTATCTGAT<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
| 0%<br />
| ACCGCAATG.....CCAGGGTGA<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
| 0%<br />
| GTCATTTAG.....TGGCATCGA<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
| 0%<br />
| CGGGGATAC.....CGTGCTGTT<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
| 0%<br />
| GTCGGGGCT.....CGGCCTGGC<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
| -44.29%<br />
| GAAGTCATG.....CGCCGTTAT<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
| 0%<br />
| TCCGAATGA.....CGTGTTTTG<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
| 0%<br />
| TGATGATCA.....TGTTCTGGC<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
| 0%<br />
| TAAAGTGAT.....CGAGGTCAC<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
| -44.27%<br />
| CCTTGCCGC.....TCCTTCATA<br />
|-<br />
| mamR<br />
| 8.04<br />
| 8.04<br />
| 0%<br />
| TCTCAACCA.....CGTGCAGGG<br />
|-<br />
| mamB<br />
| 16.56<br />
| 16.56<br />
| 0%<br />
| TCCAATCTT.....TGGGCCTTT<br />
|-<br />
| mamS<br />
| 32.86<br />
| 25.09<br />
| -23.65%<br />
| CACGGGCCT.....GATGGCCGA<br />
|-<br />
| mamT<br />
| 81.16<br />
| 81.16<br />
| 0%<br />
| TGGTGCAGT.....CTTGGGGAT<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
| 0%<br />
| CGCTGACCA.....CGCCTGCCT<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
| 0%<br />
| AATAAACCA.....TCGTGACCG<br />
|}.<br />
Using the RBS Calculator developed by the Salis, Mirsky, and Voigt ([https://salis.psu.edu/software/doReverseRBS Link]), we were able to estimate the relative translation rate of each gene from the mamAB operon. We tested this against the anti-Shine-Dalgarno 16S rRNA regions from E. coli and AMB-1, which differ only by one base – ACCTCCTTA and ACCTCCTTT, respectively. Furthermore, by modifying the native sequences, the translation initiation rates could be changed to get the proper expression levels.<br />
Each number in the table represents the translation initiation rate (au) on a proportional scale from 0.1 to 100,00+. The higher the number the more proteins are translated per mRNA. It is important to keep in mind that this RBS calculator gives an estimation of translational efficiency only.<br />
Due to the similarity in 16S rRNA sequences between the two strains, we expected to see a similar translation rates in E. coli compared with AMB-1 and the predictions fall within those expectations. However, mamN, mamQ, and mamS may have significantly lower translation rates in E. coli than AMB-1. <br />
With the use the RBS calculator, the activity of individual genes’ can be estimated thus the level gene expression can be varied by sequence modification.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-23T22:16:11Z<p>Saushun: /* sfGFP-MamK: Scaffold formation */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-23T22:11:03Z<p>Saushun: /* sfGFP-MamK: Scaffold formation */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E. coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of the mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-23T22:09:54Z<p>Saushun: /* sfGFP-MamK: Scaffold formation */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
With the presence of [http://en.wikipedia.org/wiki/IPTG IPTG], transcription of lac operon was triggered hence the expression of the mamK. On the other hand, in the absence of IPTG, the production of mamK filament was not observed. <br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ToolkitTeam:Washington/Magnetosomes/Magnet Toolkit2011-10-23T21:56:29Z<p>Saushun: /* What are magnetosomes? Where do they come from? */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Magnetosomes'''</big></big></big></big></center><br><br><br />
<br />
===What are magnetosomes? Where do they come from?===<br />
<br />
[[File:Washington iGEM2011 magnetotatic bacteria picture.jpg|thumb|right|350px|Magnetotactic Bacteria (left) and Magnetosome chains (right)]]<br />
<br />
<br> Magnetotactic bacteria are prokaryotic organisms that possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome. Magnetosomes are small invaginations of the bacterial inner membrane that contain magnetite particles.<br />
<br />
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 sense the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, magnetosomes are thought to help aid the organism in its search for the optimal oxygen level from a search in three dimensional space (in all directions) to a one dimensional space along a single path.<br />
<br><br><br><br />
<center>Video demonstration of Magnetic property of AMB-1:</center><br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/1qWK_BZS-CU" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br> <br><br />
<br />
===A Closer look at Magnetosome Formation ===<br />
<br />
[[File:F6.medium.png|300px|thumb|right|Diagram of stepwise magnetosome construction within AMB-1]]<br />
<br> <br />
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: <br />
# Membrane invagination<br />
# Acquiring minerals for magnetite formation <br />
# Iron-oxidation and reduction<br />
# Magnetite nucleation and morphology regulation.<br />
<br />
<br> Earlier gene products must be present for later gene products to be formed as shown in the diagram on the right[http://www.pnas.org/content/107/12/5593.full.pdf+html]:<br />
<br />
<br><br><br> <br />
<br />
===What did the UW iGEM team do with Magnetotactic Bacteria?=== <br />
<br> <br />
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 the only cluster essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].<br />
<br />
The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in ''E. coli''. We are doing this to learn 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 in ''E.coli''.<br />
[[File:MamAB.png|center|500px|thumb|Fig. 3: The mamAB operon (R5) located in the magnetosome island (MAI).]]<br />
<br><br />
==='''About the Magnetosome Toolkit'''===<br />
<br><br />
Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team created the '''"Magnetosome Toolkit"''' which contains many of the genes required for magnetosome formation. Providing this toolkit to the Parts Registry will help allow future iGEM teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in multiple organisms. <br />
<br> <br/><br />
As previously noted, magnetosome formation within the host-organism, ''Magnetospirillium magneticum'', strain AMB-1, is a highly regulated step-wise process. As shown in diagram of stepwise magnetosome construction above, some genes encode proteins that form an invagination of the inner membrane, other genes which help align the magnetosomes into their characteristics chains, and others which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment since they are the essential foundation for magnetosome development. In addition, the creation of a scaffold to which other genes localize is highly applicable to systems in synthetic biology. (for more information, please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Future Future Directions] page)<br />
<br><br />
<br />
The genes we focused on are <i>mamK</i> and <i>mamI</i> since they have known 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 when lost inhibits vesicle formation. <br />
(for other gene functions, please see the iGEM Toolkits parts submitted page)<br />
<br> <br><br />
<br />
==='''Toolkit Contruction'''===<br />
[[File:Washington Methode image.jpg|700px|center]]<br />
<br />
<br/>Please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results Result Summary] page for to see how far we were able to get this summer!<br />
<br />
<br/><br />
<br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ToolkitTeam:Washington/Magnetosomes/Magnet Toolkit2011-10-23T21:52:43Z<p>Saushun: /* What are magnetosomes? Where do they come from? */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Magnetosomes'''</big></big></big></big></center><br><br><br />
<br />
===What are magnetosomes? Where do they come from?===<br />
<br />
[[File:Washington iGEM2011 magnetotatic bacteria picture.jpg|thumb|right|350px|Magnetotactic Bacteria (left) and Magnetosome chains (right)]]<br />
<br />
<br> Magnetotactic bacteria are prokaryotic organisms that possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome. Magnetosomes are small invaginations of the bacterial inner membrane that contain magnetite particles.<br />
<br />
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 sense the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, magnetosomes are thought to help aid the organism in its search for the optimal oxygen level from a search in three dimensional space (in all directions) to a one dimensional space along a single path.<br />
<br />
Video demonstration of Magnetic property of AMB-1:<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/1qWK_BZS-CU" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br> <br><br />
<br />
===A Closer look at Magnetosome Formation ===<br />
<br />
[[File:F6.medium.png|300px|thumb|right|Diagram of stepwise magnetosome construction within AMB-1]]<br />
<br> <br />
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: <br />
# Membrane invagination<br />
# Acquiring minerals for magnetite formation <br />
# Iron-oxidation and reduction<br />
# Magnetite nucleation and morphology regulation.<br />
<br />
<br> Earlier gene products must be present for later gene products to be formed as shown in the diagram on the right[http://www.pnas.org/content/107/12/5593.full.pdf+html]:<br />
<br />
<br><br><br> <br />
<br />
===What did the UW iGEM team do with Magnetotactic Bacteria?=== <br />
<br> <br />
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 the only cluster essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].<br />
<br />
The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in ''E. coli''. We are doing this to learn 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 in ''E.coli''.<br />
[[File:MamAB.png|center|500px|thumb|Fig. 3: The mamAB operon (R5) located in the magnetosome island (MAI).]]<br />
<br><br />
==='''About the Magnetosome Toolkit'''===<br />
<br><br />
Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team created the '''"Magnetosome Toolkit"''' which contains many of the genes required for magnetosome formation. Providing this toolkit to the Parts Registry will help allow future iGEM teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in multiple organisms. <br />
<br> <br/><br />
As previously noted, magnetosome formation within the host-organism, ''Magnetospirillium magneticum'', strain AMB-1, is a highly regulated step-wise process. As shown in diagram of stepwise magnetosome construction above, some genes encode proteins that form an invagination of the inner membrane, other genes which help align the magnetosomes into their characteristics chains, and others which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment since they are the essential foundation for magnetosome development. In addition, the creation of a scaffold to which other genes localize is highly applicable to systems in synthetic biology. (for more information, please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Future Future Directions] page)<br />
<br><br />
<br />
The genes we focused on are <i>mamK</i> and <i>mamI</i> since they have known 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 when lost inhibits vesicle formation. <br />
(for other gene functions, please see the iGEM Toolkits parts submitted page)<br />
<br> <br><br />
<br />
==='''Toolkit Contruction'''===<br />
[[File:Washington Methode image.jpg|700px|center]]<br />
<br />
<br/>Please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results Result Summary] page for to see how far we were able to get this summer!<br />
<br />
<br/><br />
<br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-23T21:48:08Z<p>Saushun: /* sfGFP-MamK: Scaffold formation */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing ''E coli'':<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-22T02:21:49Z<p>Saushun: /* What’s in the Magnetosome Toolkit? */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing E coli:<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-22T02:21:32Z<p>Saushun: /* Table of computationally predicted cloning efficiencies */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing E coli:<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-21T22:25:22Z<p>Saushun: /* Table of computationally predicted cloning efficiencies */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing E coli:<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
=='''Table of computationally predicted cloning efficiencies ''' == <br />
The following results and analyses are preliminary. <br />
<br />
The following is a list of computationally predicted cloning efficiencies for each gene we cloned, from the publicly available [https://salis.psu.edu/software/RBSLibraryCalculatorSearchMode RBS Calculator]. <br />
<br />
{| class="wikitable"<br />
|-<br />
! mam gene<br />
! AMB-1<br />
! ''E.Coli''<br />
|-<br />
| mamH<br />
| 10821.63<br />
| 10821.63<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
|-<br />
| mamE<br />
| 1.59<br />
| 1.62<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
|-<br />
| mamR<br />
| XXX<br />
| XXX<br />
|- <br />
| mamB<br />
| 16.56<br />
| 16.56<br />
|- <br />
| mamS<br />
| 32.86<br />
| 25.09<br />
|- <br />
| mamT<br />
| 81.16<br />
| 81.16<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
<br />
|}.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-21T22:02:50Z<p>Saushun: /* A table of cloning efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
Here is a video showing the filaments connecting the growing E coli:<br />
<br />
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html><br />
<br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
=='''A table of cloning efficiency (DRAFT) ''' == <br />
This part of results and analysis is NOT finalized. <br />
<br />
The following is a list of computationally predicted cloning efficiencies for each gene we cloned, from the RBS tool available at http://rbs.location.org. <br />
<br />
{| class="wikitable"<br />
|-<br />
! mam gene<br />
! AMB-1<br />
! ''E.Coli''<br />
|-<br />
| mamH<br />
| 10821.63<br />
| 10821.63<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
|-<br />
| mamE<br />
| 1.59<br />
| 1.62<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
|-<br />
| mamR<br />
| XXX<br />
| XXX<br />
|- <br />
| mamB<br />
| 16.56<br />
| 16.56<br />
|- <br />
| mamS<br />
| 32.86<br />
| 25.09<br />
|- <br />
| mamT<br />
| 81.16<br />
| 81.16<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
<br />
|}.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ToolkitTeam:Washington/Magnetosomes/Magnet Toolkit2011-10-21T20:13:41Z<p>Saushun: /* What are magnetosomes? Where do they come from? */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Magnetosomes'''</big></big></big></big></center><br><br><br />
<br />
===What are magnetosomes? Where do they come from?===<br />
<br />
[[File:Washington iGEM2011 magnetotatic bacteria picture.jpg|thumb|right|350px|Magnetotactic Bacteria (left) and Magnetosome chains (right)]]<br />
<br />
<br> Magnetotactic bacteria are prokaryotic organisms that possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome. Magnetosomes are small invaginations of the bacterial inner membrane that contain magnetite particles.<br />
<br />
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 sense the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, magnetosomes are thought to help aid the organism in its search for the optimal oxygen level from a search in three dimensional space (in all directions) to a one dimensional space along a single path.<br />
<br />
[http://youtu.be/1qWK_BZS-CU Video demonstration of Magnetic property of AMB-1 ]<br />
<br />
<br> <br><br />
<br />
===A Closer look at Magnetosome Formation ===<br />
<br />
[[File:F6.medium.png|300px|thumb|right|Diagram of stepwise magnetosome construction within AMB-1]]<br />
<br> <br />
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: <br />
# Membrane invagination<br />
# Acquiring minerals for magnetite formation <br />
# Iron-oxidation and reduction<br />
# Magnetite nucleation and morphology regulation.<br />
<br />
<br> Earlier gene products must be present for later gene products to be formed as shown in the diagram on the right[http://www.pnas.org/content/107/12/5593.full.pdf+html]:<br />
<br />
<br><br><br> <br />
<br />
===What did the UW iGEM team do with Magnetotactic Bacteria?=== <br />
<br> <br />
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 the only cluster essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].<br />
<br />
The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in ''E. coli''. We are doing this to learn 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 in ''E.coli''.<br />
[[File:MamAB.png|center|500px|thumb|Fig. 3: The mamAB operon (R5) located in the magnetosome island (MAI).]]<br />
<br><br />
==='''About the Magnetosome Toolkit'''===<br />
<br><br />
Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team created the '''"Magnetosome Toolkit"''' which contains many of the genes required for magnetosome formation. Providing this toolkit to the Parts Registry will help allow future iGEM teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in multiple organisms. <br />
<br> <br/><br />
As previously noted, magnetosome formation within the host-organism, ''Magnetospirillium magneticum'', strain AMB-1, is a highly regulated step-wise process. As shown in diagram of stepwise magnetosome construction above, some genes encode proteins that form an invagination of the inner membrane, other genes which help align the magnetosomes into their characteristics chains, and others which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment since they are the essential foundation for magnetosome development. In addition, the creation of a scaffold to which other genes localize is highly applicable to systems in synthetic biology. (for more information, please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Future Future Directions] page)<br />
<br><br />
<br />
The genes we focused on are <i>mamK</i> and <i>mamI</i> since they have known 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 when lost inhibits vesicle formation. <br />
(for other gene functions, please see the iGEM Toolkits parts submitted page)<br />
<br> <br><br />
<br />
==='''Toolkit Contruction'''===<br />
[[File:Washington Methode image.jpg|700px|center]]<br />
<br />
<br/>Please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results Result Summary] page for to see how far we were able to get this summer!<br />
<br />
<br/><br />
<br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ToolkitTeam:Washington/Magnetosomes/Magnet Toolkit2011-10-21T19:41:40Z<p>Saushun: /* What are magnetosomes? Where do they come from? */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Magnetosomes'''</big></big></big></big></center><br><br><br />
<br />
===What are magnetosomes? Where do they come from?===<br />
<br />
[[File:Washington iGEM2011 magnetotatic bacteria picture.jpg|thumb|right|350px|Magnetotactic Bacteria (left) and Magnetosome chains (right)]]<br />
<br />
<br> Magnetotactic bacteria are prokaryotic organisms that possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome. Magnetosomes are small invaginations of the bacterial inner membrane that contain magnetite particles.<br />
<br />
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 sense the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, magnetosomes are thought to help aid the organism in its search for the optimal oxygen level from a search in three dimensional space (in all directions) to a one dimensional space along a single path.<br />
<br />
[[Media:http://youtu.be/1qWK_BZS-CU]]<br />
<br />
<br> <br><br />
<br />
===A Closer look at Magnetosome Formation ===<br />
<br />
[[File:F6.medium.png|300px|thumb|right|Diagram of stepwise magnetosome construction within AMB-1]]<br />
<br> <br />
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: <br />
# Membrane invagination<br />
# Acquiring minerals for magnetite formation <br />
# Iron-oxidation and reduction<br />
# Magnetite nucleation and morphology regulation.<br />
<br />
<br> Earlier gene products must be present for later gene products to be formed as shown in the diagram on the right[http://www.pnas.org/content/107/12/5593.full.pdf+html]:<br />
<br />
<br><br><br> <br />
<br />
===What did the UW iGEM team do with Magnetotactic Bacteria?=== <br />
<br> <br />
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 the only cluster essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].<br />
<br />
The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in ''E. coli''. We are doing this to learn 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 in ''E.coli''.<br />
[[File:MamAB.png|center|500px|thumb|Fig. 3: The mamAB operon (R5) located in the magnetosome island (MAI).]]<br />
<br><br />
==='''About the Magnetosome Toolkit'''===<br />
<br><br />
Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team created the '''"Magnetosome Toolkit"''' which contains many of the genes required for magnetosome formation. Providing this toolkit to the Parts Registry will help allow future iGEM teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in multiple organisms. <br />
<br> <br/><br />
As previously noted, magnetosome formation within the host-organism, ''Magnetospirillium magneticum'', strain AMB-1, is a highly regulated step-wise process. As shown in diagram of stepwise magnetosome construction above, some genes encode proteins that form an invagination of the inner membrane, other genes which help align the magnetosomes into their characteristics chains, and others which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment since they are the essential foundation for magnetosome development. In addition, the creation of a scaffold to which other genes localize is highly applicable to systems in synthetic biology. (for more information, please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Future Future Directions] page)<br />
<br><br />
<br />
The genes we focused on are <i>mamK</i> and <i>mamI</i> since they have known 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 when lost inhibits vesicle formation. <br />
(for other gene functions, please see the iGEM Toolkits parts submitted page)<br />
<br> <br><br />
<br />
==='''Toolkit Contruction'''===<br />
[[File:Washington Methode image.jpg|700px|center]]<br />
<br />
<br/>Please see our [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results Result Summary] page for to see how far we were able to get this summer!<br />
<br />
<br/><br />
<br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-21T19:14:24Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].<br />
<br />
=='''A table of cloning efficiency ''' ==<br />
{| class="wikitable"<br />
|-<br />
! mam gene<br />
! AMB-1<br />
! ''E.Coli''<br />
|-<br />
| mamH<br />
| 10821.63<br />
| 10821.63<br />
|-<br />
| mamI<br />
| 88.93<br />
| 88.93<br />
|-<br />
| mamE<br />
| 1.59<br />
| 1.62<br />
|-<br />
| mamJ<br />
| 105.9<br />
| 105.9<br />
|- <br />
| mamK<br />
| 1056.92<br />
| 1056.92<br />
|-<br />
| mamL<br />
| 584.21<br />
| 584.21<br />
|-<br />
| mamM<br />
| 930.1<br />
| 930.1<br />
|-<br />
| mamN<br />
| 413.73<br />
| 230.48<br />
|- <br />
| mamO<br />
| 138.73<br />
| 138.73<br />
|-<br />
| mamP<br />
| 198.85<br />
| 198.85<br />
|-<br />
| mamA<br />
| 14.29<br />
| 14.29<br />
|-<br />
| mamQ<br />
| 22.41<br />
| 12.49<br />
|-<br />
| mamR<br />
| XXX<br />
| XXX<br />
|- <br />
| mamB<br />
| 16.56<br />
| 16.56<br />
|- <br />
| mamS<br />
| 32.86<br />
| 25.09<br />
|- <br />
| mamT<br />
| 81.16<br />
| 81.16<br />
|- <br />
| mamU<br />
| 33.58<br />
| 33.58<br />
|- <br />
| mamV<br />
| 1.61<br />
| 1.61<br />
<br />
|}.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-10-21T18:57:38Z<p>Saushun: /* What’s in the Magnetosome Toolkit? */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
* A table of cloning efficiency <br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-29T02:03:58Z<p>Saushun: /* sfGFP-MamI: Membrane localization */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1<br />
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
-----<br><br />
<br />
== '''Superfolder GFP-magnetosome gene protein fusions'''==<br />
<br />
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 [https://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.'' <br />
<br />
===sfGFP-MamK: Scaffold formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br><br />
<br />
===sfGFP-MamI: Membrane localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For <i>mamI</i>, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. The membrane localization 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br/><br/><br />
<br />
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}.<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectorsTeam:Washington/Magnetosomes/GibsonVectors2011-09-29T00:24:23Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Gibson Assembly Vectors'''</big></big></big></big></center><br><br><br />
=== About Gibson Assembly===<br />
Gibson assembly is a new synthetic biology tool that allows scar-free assembly of multiple gene-inserts in one isothermal reaction by using an exonuclease, polymerase, and heat-stable ligase to chew back, anneal, and repair gaps from homologous DNA fragments. This method is gaining popularity as it tends to be more efficient than standard restriction/ligation assembly, which can save a great amount of time in the cloning process. Overall, Gibson assembly allows teams to built large gene constructs with ease. <br />
<br/> [[File:Igem2011_GibsonReacion.png|center|400px]] <br />
<br><br><br />
-------------<br />
<br><br />
<br />
=== What happened last year?===<br />
The Gibson Cloning method is definitely not a new method to be introduced to the iGEM community. <br />
In 2010, the Cambridge iGEM team created the [http://www.cambridgeigem.org/RFC57.pdf BBF RFC 57] document which outlines a protocol for Gibson Assembly using standard BioBricks ([http://dspace.mit.edu/bitstream/handle/1721.1/45138/BBFRFC10.txt?sequence=1 BBF RFC 10]) that would allow many fragment inserts during a single cloning step. However, while creating the Magnetosome Toolkit, we found that this BioBrick standard was incapable of producing high yields of desired Gibson products even for two-fragment assemblies.<br />
<br><br />
<br />
The "pSB" standard BioBrick vectors available through iGEM are not designed for efficient multiple-insert cloning beyond [http://partsregistry.org/Assembly:3A_Assembly three fragments] and are limited by ligation scars. The primary problem with a standard pSB vector is the self-complementarity of the two NotI sequences embedded in both the BioBrick prefix and suffix. These sequences prevent gene inserts from being incorporated efficiently, and do not produce a high yield of the Gibson product even in two-fragment assemblies. Generally, the backbone self-anneals and the recircularized plasmid has a combined prefix and suffix region that reads EcoRI-NotI-PstI. <br/><br />
[[File:Igem2011 biobrick NotI.png|600px|center]]<br />
<br />
<br />
<br />
To overcome this problem, the [https://2010.igem.org/Team:Washington/Tools_Used/Next-Gen_Cloning 2010 UW iGEM] team developed new prefix and suffix regions that are based on BglBrick (BBF [http://dspace.mit.edu/handle/1721.1/46747 RFC 21]) standard and designed to eliminate self-complementarity from the prefix and suffix of the plasmid. These vectors [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults dramatically] increase the Gibson assembly efficiency of large-scale gene assemblies and are also compatible with iGEM standard BioBrick parts. <br/><br />
[[File:Igem2011_gibsonbrick.png|600px|center]]<br />
<br />
<br><br />
<br />
=== What did we do this year?===<br />
Seeing that this is a very efficient method to do cloning, we continued to make improvements to the methods and created a '''Gibson Assembly Toolkit'''! <br> <br/><br />
<br />
[[File:Washington_iGEM2011_how_to_make_vector.png|right|thumb|450px]]<br />
<br />
===Creation of 5 plasmid vectors===<br />
Our new vectors for Gibson assembly follow the naming convention of pGA. To make our pGA vectors, we first amplified the backbones and the pLac GFP insert respectively. Then we performed a Gibson reaction to combine them together to make the pGA vectors. <br />
<br />
All togeher, we created 5 pGA vectors and submitted them to the registry:<br />
* 2 High copy extraction/cloning vectors<br />
** pGA1A3, pGA1C3<br />
* 1 medium copy expression vector<br />
** pGA3K3<br />
* 2 low copy expression vectors<br />
** pGA4A5, pGA4C5<br />
<br />
As listed above, each of our vectors have varying copy numbers, antibiotic resistances, and purposes within the magnetosome gene assembly. However, they all appear to be very efficient will multiple gene inserts.<br />
<br/><br/><br/><br/><br/><br />
For experimental details comparing the efficiencies of pSB1A3 and pGA1A3, see [https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults our assay results]. In addition to the characterization of assembly efficiency, we used the pGA vectors for the [https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome Toolkit] project. First, we efficiently extracted and BioBricked all the 18 <i>mamAB</i> magnetosome genes. We also made super-assemblies from these BioBricks to make 10 kilobase plasmids (see [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590017 mamHIEJKL] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590019 mamQRBSTUV]) from 2 kilobase pieces by assembling up to five fragments all in one cloning step! <br />
<br />
<br />
<br />
====References====<br />
<br />
1. Daniel G Gibson, Lei Young, Ray-Yuan Chuang, Craig J Venter, Clyde A Hutchison, and Hamilton O Smith. Enzymatic assembly of DNA molecules up to several hundred kilobases. <i>Nat Methods</i>, <b>6</b>(5):343, Apr 2009.<br><br />
2. Peter A Carr and George M Church. Genome Engineering. <i>Nat Biotechnol</i> , <b>27</b>(12):1151 Dec 2009.<br><br />
3. Daniel G Gibson, Hamilton O Smith, Clyde A Hutchison Iii, J Craig Venter, and<br />
Chuck Merryman. Chemical synthesis of the mouse mitochondrial genome. <i>Nat Methods</i>, <b>7</b>(11):901, Oct 2010.</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/PartsTeam:Washington/Magnetosomes/Parts2011-09-28T23:00:12Z<p>Saushun: /* Description of mamAB operon gene functions */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Parts Submitted'''</big></big></big></big></center><br><br><br />
<br />
The iGEM toolkits group submitted a total twenty-two parts to the registry: <br />
* 10 magnetosome gene groups from <i>mamAB</i> operon<br />
* 3 superassemblies of the <i>mamAB</i> operon from the set of 10 gene groups<br />
* 2 <i>sfGFP-mamK</i> and <i>sfGFP-mamI</i> gene fusions<br />
* 5 Gibson Assembly (pGA) vectors<br />
<br/><br />
=='''Magnetosome Toolkit Favorite Parts'''==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015 '''sfGFP_mamK_pGA1C3''']<br />
<br />
This part consists of the <i>mamK</i> gene from <i>Magnetospirillum magneticum</i> strain AMB-1, fused to superfolder <i>gfp</i> on pGA1C3. MamK has been reported to be essential for proper magnetosome formation in magnetotactic bacteria; its bacterial actin-like cytoskeleton protein is required for proper alignment of the magnetosomes in a chain. Beyond magnetosome alignment, the MamK filament could act as a scaffold for protein localization or to alter cell morphology.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3''']<br />
<br />
This part consists of the <i>mamI</i> gene from <i>Magnetospirillum magneticum</i> strain AMB-1, fused to superfolder <i>gfp</i> on pGA1C3. MamI is a membrane-localized protein that is essential for magnetosome vesicle formation, and is also known to bind the MamK filament. Since it localizes to the membrane, MamI may be useful in maximizing the flux of biosynthetic pathways as a membrane-linker protein.<br />
<br />
=='''The Gibson Assembly Toolkit'''==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010 '''pGA1A3_pLacGFP''']<br />
<br />
This is a high copy plasmid backbone (replication origin pMB1) that confers ampicillin resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB1A3 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011 '''pGA1C3_pLacGFP''']<br />
<br />
This is a high copy plasmid backbone (replication origin pMB1) that confers chloramphenicol resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB1C3 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014 '''pGA3K3_pLacGFP''']<br />
<br />
This is a medium copy plasmid backbone (replication origin p15A) that confers kanamycin resistance. It has an insert with a LacI-repressible promoter driving GFP expression. pGA3K3 is identical to pSB3K3 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012 '''pGA4C5_pLacGFP''']<br />
<br />
This is a low copy plasmid backbone (replication origin pSC101) that confers chloramphenicol resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB4C5 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013 '''pGA4A5_pLacGFP''']<br />
<br />
This is a low copy plasmid backbone (replication origin pSC101) that confers ampicillin resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB4A5 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly. It is also notable that though we searched in multiple BioBrick distributions from different years, we could not find any that could be sequence-verified as pSB4A5 (the Vf2 sequence reads all suggested pSB1A2). We then generated pGA4A5 from pGA4C5 using the ampicillin cassette from pSB1A3.<br />
<br />
====Description of <i>mamAB</i> operon gene functions====<br />
<br />
{| class="wikitable"<br />
|-<br />
! Gene <br />
! AMB Number<br />
! Cluster Membership<br />
! Member of 28 genes list? (specific*/related**)<br />
! Function Summary (Vesicle chain formation, and/or biomineralization)<br />
! Gene Function<br />
|-<br />
| mamH<br />
| amb0961<br />
| mamAB<br />
| Related<br />
| unknown<br />
| N/A<br />
|-<br />
| mamI<br />
| amb0962<br />
| mamAB<br />
| Specific<br />
| Vesicle, Chain Formation<br />
| >Loss of mamI causes no membrane formation, gene product is localized onto chains<br />
|-<br />
| mamE<br />
| amb0963<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >Membrane-bound serine protease required for magnetite formation; might control the localization of other magnetosome proteins<br />
|-<br />
| mamJ<br />
| amb0964<br />
| mamAB; mam Islet<br />
| Specific<br />
| Chain Formation<br />
| >Proper magnetosome chain organization/assembly<br />
|-<br />
| mamK<br />
| amb0965<br />
| mamAB; mam Islet<br />
| Related<br />
| Chain Formation<br />
| >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<br />
|- <br />
| mamL<br />
| amb0966<br />
| mamAB; mam Islet<br />
| Specific<br />
| Vesicle, biomineralization<br />
| >Crucial to mangneosome membrane creation, shown to be spread across the cell membrane and sometimes forms lines<br />
|-<br />
| mamM<br />
| amb0967<br />
| mamAB<br />
| Related<br />
|<br />
| >Biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamN<br />
| amb0968<br />
| mamAB<br />
| Related<br />
|<br />
| >Biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamO<br />
| amb0969<br />
| mamAB<br />
| Related<br />
|<br />
| >Biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|- <br />
| mamP<br />
| amb0970<br />
| mamAB<br />
| Related<br />
| Biomineralization<br />
| >Loss of mamP causes weak magnetic response, with large but fewer crystals<br />
|-<br />
| mamA<br />
| amb0971<br />
| mamAB<br />
| Related<br />
|<br />
| >Required for magnetosome activation; activation of vessicles<br />
|-<br />
| mamQ<br />
| amb0972<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >ORF; formation/maintenance of magnetosome membranes<br />
|-<br />
| mamR<br />
| amb0973<br />
| mamAB<br />
| Specific<br />
| Chain formation, Biomineralization<br />
| >ORF; plays a role in controlling both particle number and size of magnetite cyrstals<br />
|-<br />
| mamB<br />
| amb0974<br />
| mamAB<br />
| Related<br />
| Vesicle, Biomineralization<br />
| >Indirect role in magnetosome membrane invagination and biomineralization; magnetosome compartment formation<br />
|-<br />
| mamS<br />
| amb0975<br />
| mamAB<br />
| Specific<br />
| unknown function<br />
| N/A<br />
|-<br />
| mamT<br />
| amb0976<br />
| mamAB<br />
| Specific<br />
| Biomineralization<br />
| >Magnetite crystal growth; participates in different steps during magnetite synthesis<br />
|-<br />
| mamU<br />
| amb0977<br />
| mamAB<br />
| Related<br />
| unknown function<br />
| N/A<br />
|-<br />
| mamV<br />
| amb0978<br />
| mamAB<br />
| unknown function<br />
| N/A<br />
| N/A<br />
|-<br />
|}</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/PartsTeam:Washington/Magnetosomes/Parts2011-09-28T22:57:24Z<p>Saushun: /* Description of mamAB operon gene functions */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Parts Submitted'''</big></big></big></big></center><br><br><br />
<br />
The iGEM toolkits group submitted a total twenty-two parts to the registry: <br />
* 10 magnetosome gene groups from <i>mamAB</i> operon<br />
* 3 superassemblies of the <i>mamAB</i> operon from the set of 10 gene groups<br />
* 2 <i>sfGFP-mamK</i> and <i>sfGFP-mamI</i> gene fusions<br />
* 5 Gibson Assembly (pGA) vectors<br />
<br/><br />
=='''Magnetosome Toolkit Favorite Parts'''==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015 '''sfGFP_mamK_pGA1C3''']<br />
<br />
This part consists of the <i>mamK</i> gene from <i>Magnetospirillum magneticum</i> strain AMB-1, fused to superfolder <i>gfp</i> on pGA1C3. MamK has been reported to be essential for proper magnetosome formation in magnetotactic bacteria; its bacterial actin-like cytoskeleton protein is required for proper alignment of the magnetosomes in a chain. Beyond magnetosome alignment, the MamK filament could act as a scaffold for protein localization or to alter cell morphology.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3''']<br />
<br />
This part consists of the <i>mamI</i> gene from <i>Magnetospirillum magneticum</i> strain AMB-1, fused to superfolder <i>gfp</i> on pGA1C3. MamI is a membrane-localized protein that is essential for magnetosome vesicle formation, and is also known to bind the MamK filament. Since it localizes to the membrane, MamI may be useful in maximizing the flux of biosynthetic pathways as a membrane-linker protein.<br />
<br />
=='''The Gibson Assembly Toolkit'''==<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010 '''pGA1A3_pLacGFP''']<br />
<br />
This is a high copy plasmid backbone (replication origin pMB1) that confers ampicillin resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB1A3 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011 '''pGA1C3_pLacGFP''']<br />
<br />
This is a high copy plasmid backbone (replication origin pMB1) that confers chloramphenicol resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB1C3 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014 '''pGA3K3_pLacGFP''']<br />
<br />
This is a medium copy plasmid backbone (replication origin p15A) that confers kanamycin resistance. It has an insert with a LacI-repressible promoter driving GFP expression. pGA3K3 is identical to pSB3K3 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012 '''pGA4C5_pLacGFP''']<br />
<br />
This is a low copy plasmid backbone (replication origin pSC101) that confers chloramphenicol resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB4C5 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013 '''pGA4A5_pLacGFP''']<br />
<br />
This is a low copy plasmid backbone (replication origin pSC101) that confers ampicillin resistance. It has an insert with a LacI-repressible promoter driving GFP expression. It is identical to pSB4A5 except for its bglBrick prefix and suffix, and is optimized for Gibson Assembly. It is also notable that though we searched in multiple BioBrick distributions from different years, we could not find any that could be sequence-verified as pSB4A5 (the Vf2 sequence reads all suggested pSB1A2). We then generated pGA4A5 from pGA4C5 using the ampicillin cassette from pSB1A3.<br />
<br />
====Description of <i>mamAB</i> operon gene functions====<br />
<br />
{| class="wikitable"<br />
|-<br />
! Gene <br />
! AMB Number<br />
! Cluster Membership<br />
! Member of 28 genes list? (specific*/related**)<br />
! Function Summary (Vesicle chain formation, and/or biomineralization)<br />
! Gene Function<br />
|-<br />
| mamH<br />
| amb0961<br />
| mamAB<br />
| Related<br />
|<br />
|<br />
|-<br />
| mamI<br />
| amb0962<br />
| mamAB<br />
| Specific<br />
| Vesicle, Chain Formation<br />
| >Loss of mamI causes no membrane formation, gene product is localized onto chains<br />
|-<br />
| mamE<br />
| amb0963<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >Membrane-bound serine protease required for magnetite formation; might control the localization of other magnetosome proteins<br />
|-<br />
| mamJ<br />
| amb0964<br />
| mamAB; mam Islet<br />
| Specific<br />
| Chain Formation<br />
| >Proper magnetosome chain organization/assembly<br />
|-<br />
| mamK<br />
| amb0965<br />
| mamAB; mam Islet<br />
| Related<br />
| Chain Formation<br />
| >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<br />
|- <br />
| mamL<br />
| amb0966<br />
| mamAB; mam Islet<br />
| Specific<br />
| Vesicle, biomineralization<br />
| >Crucial to mangneosome membrane creation, shown to be spread across the cell membrane and sometimes forms lines<br />
|-<br />
| mamM<br />
| amb0967<br />
| mamAB<br />
| Related<br />
|<br />
| >Biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamN<br />
| amb0968<br />
| mamAB<br />
| Related<br />
|<br />
| >Biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|-<br />
| mamO<br />
| amb0969<br />
| mamAB<br />
| Related<br />
|<br />
| >Biomineralization, involved in iron transport, magnetite nucleation, or establishement of the proper chemical enviornment for magnetite synthesis in the magnetosome<br />
|- <br />
| mamP<br />
| amb0970<br />
| mamAB<br />
| Related<br />
| Biomineralization<br />
| >Loss of mamP causes weak magnetic response, with large but fewer crystals<br />
|-<br />
| mamA<br />
| amb0971<br />
| mamAB<br />
| Related<br />
|<br />
| >Required for magnetosome activation; activation of vessicles<br />
|-<br />
| mamQ<br />
| amb0972<br />
| mamAB; mam Islet<br />
| Related<br />
|<br />
| >ORF; formation/maintenance of magnetosome membranes<br />
|-<br />
| mamR<br />
| amb0973<br />
| mamAB<br />
| Specific<br />
| Chain formation, Biomineralization<br />
| >ORF; plays a role in controlling both particle number and size of magnetite cyrstals<br />
|-<br />
| mamB<br />
| amb0974<br />
| mamAB<br />
| Related<br />
| Vesicle, Biomineralization<br />
| >Indirect role in magnetosome membrane invagination and biomineralization; magnetosome compartment formation<br />
|-<br />
| mamS<br />
| amb0975<br />
| mamAB<br />
| Specific<br />
| unknown function<br />
| N/A<br />
|-<br />
| mamT<br />
| amb0976<br />
| mamAB<br />
| Specific<br />
| Biomineralization<br />
| >Magnetite crystal growth; participates in different steps during magnetite synthesis<br />
|-<br />
| mamU<br />
| amb0977<br />
| mamAB<br />
| Related<br />
| unknown function<br />
| N/A<br />
|-<br />
| mamV<br />
| amb0978<br />
| mamAB<br />
| unknown function<br />
| N/A<br />
|<br />
|-<br />
|}</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:47:45Z<p>Saushun: /* Construction of the R5 region of the Magnetosome Island in E.coli */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors <br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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. <br />
(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.'' <br />
<br />
===mamK: Filament formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br />
===mamI: Membrane Localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
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/><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]]<br />
</center><br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:46:23Z<p>Saushun: /* Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors <br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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. <br />
(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.'' <br />
<br />
===mamK: Filament formation=== <br />
<br><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br />
===mamI: Membrane Localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:40:25Z<p>Saushun: /* mamK: Filament formation */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors <br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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. <br />
(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.'' <br />
<br />
<center>===mamK: Filament formation===</center><br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI: Membrane Localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:39:38Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors <br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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. <br />
(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.'' <br />
<br />
===mamK: Filament formation===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI: Membrane Localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:35:43Z<p>Saushun: /* Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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. <br />
(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.'' <br />
<br />
===mamK Filament formation in ''E.coli''===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI-- Membrane Localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:23:40Z<p>Saushun: /* mamI */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK Filament formation in ''E.coli''===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI-- Membrane Localization===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/File:Igem2011_gibsonbrick.pngFile:Igem2011 gibsonbrick.png2011-09-28T22:17:55Z<p>Saushun: uploaded a new version of &quot;File:Igem2011 gibsonbrick.png&quot;</p>
<hr />
<div></div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T22:07:55Z<p>Saushun: /* Superfolder GFP and mamK Fusion in E.coli */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK Filament formation in ''E.coli''===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:29:46Z<p>Saushun: /* mamK */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===Superfolder GFP and mamK Fusion in ''E.coli''===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:27:41Z<p>Saushun: /* mamK */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br><br />
<br />
===mamI===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:25:18Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center><br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center><br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:24:14Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
</center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] <br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:23:42Z<p>Saushun: /* mamI */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] <br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:22:56Z<p>Saushun: /* mamI */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
<center> [[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] <br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:22:23Z<p>Saushun: /* mamI */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
[[File:Washington igem11 MamIfusion full.jpg|200px|]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] <br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T21:22:09Z<p>Saushun: /* mamI */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
[[File:Washington igem11 MamIfusion full.jpg|200px|]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|400px]] <br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/File:Washington_igem11_mamI_graph.pngFile:Washington igem11 mamI graph.png2011-09-28T21:21:31Z<p>Saushun: uploaded a new version of &quot;File:Washington igem11 mamI graph.png&quot;</p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_mamI_graph.pngFile:Washington igem11 mamI graph.png2011-09-28T21:09:56Z<p>Saushun: uploaded a new version of &quot;File:Washington igem11 mamI graph.png&quot;</p>
<hr />
<div></div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T20:08:21Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br> <br> <br />
===mamI===<br />
<br />
[[File:Washington igem11 MamIfusion full.jpg|200px|]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|500px]] <br />
<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br />
<br> <br><br />
-----<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/File:Washington_igem11_mamI_graph.pngFile:Washington igem11 mamI graph.png2011-09-28T20:02:39Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_MamIfusion_GFP.jpgFile:Washington igem11 MamIfusion GFP.jpg2011-09-28T19:50:05Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_MamIfusion_full.jpgFile:Washington igem11 MamIfusion full.jpg2011-09-28T19:49:21Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-28T19:46:53Z<p>Saushun: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
=='''What’s in the Magnetosome Toolkit?'''==<br />
<br />
* Our favorite genes in pGA vectors<br />
* A set of the 18 essential genes for the various steps of magnetosome formation<br />
* A table compiling individual gene functions from our literature search<br />
<br> <br />
----- <br/><br />
<br />
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==<br />
<br />
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 is loss inhibits membrane formation. <br />
(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.'' <br />
<br />
===mamK===<br />
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]] <br />
<br />
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 our image of mamK, a filament is 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. <br />
<br />
<gallery widths=180px heights=150px caption="Just a place holder for more cool pictures" ><br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg <br />
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg<br />
File:Washington SfGFP K 4C5-col1 03 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg<br />
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg<br />
<br />
</gallery><br />
<br />
===mamI===<br />
For mamI, the gene product is seen to fluorescent around the bacterial cell membrane of the bacteria but mostly concentrated at the ends. This is seen in the graph that was taking 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.<br />
<br> <br><br />
<br />
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, 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/><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]]<br />
</center><br />
<br />
<br />
<br />
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==<br />
<br />
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: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
<br />
== '''A table of individual gene functions ''' ==<br />
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].</div>Saushunhttp://2011.igem.org/File:Washington_igem11_SfGFP_K_4C5-col1_04_gfp.jpgFile:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg2011-09-28T19:46:21Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_SfGFP_K_4C5-col1_04_crop.jpgFile:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg2011-09-28T19:45:45Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_SfGFP_K_4C5-col1_05_gfp.jpgFile:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg2011-09-28T19:43:58Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_SfGFP_K_4C5-col1_05_crop.jpgFile:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg2011-09-28T19:43:10Z<p>Saushun: </p>
<hr />
<div></div>Saushunhttp://2011.igem.org/File:Washington_igem11_MamK_fusion_gfp_01.jpgFile:Washington igem11 MamK fusion gfp 01.jpg2011-09-28T19:39:18Z<p>Saushun: </p>
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<div></div>Saushun