Team:Washington/Magnetosomes/Magnet Results

From 2011.igem.org

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=='''What’s in the Magnetosome Toolkit?'''==
=='''What’s in the Magnetosome Toolkit?'''==
-
* Our favorite genes in pGA vectors
+
* A set of 10 gene clusters from the essential mamAB operon of strain AMB-1
-
* A set of the 18 essential genes for the various steps of magnetosome formation
+
* Our favorite genes as translational fusions with superfolder <i>gfp</i> in pGA vectors
* A table compiling individual gene functions from our literature search
* A table compiling individual gene functions from our literature search
 +
* A table of Translational Efficiency using RBS calculator
 +
<br>  
<br>  
-
----- <br/>
+
-----<br>
-
 
+
-
== '''Our favorite genes in pGA vectors: Magnetosome gene-protein Fusions'''==
+
-
 
+
-
As previously stated, our genes of interest were mamK and mamI as they have functions related to localization of the magnetosome. Specifically, mamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. mamK is also shown to localize the mamI, which is loss inhibits membrane formation.
+
-
(for other gene functions, please see the iGEM Toolkits parts submitted page)Using our two genes of interest, we created C-terminal sfGFP fusions so we could track the localization of each gene separately within ''E.coli.''
+
-
 
+
-
[[File:Washington_Igem2011_mamK_and_I.png|700px|center]]
+
-
 
+
-
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''. In the image of  mamK, a filament is seen running through the length of many bacteria. For mamI, the gene product is seen to fluoresce around the cell membrane of the bacteria but mostly concentrated at the ends. Similarly, the graph shows that as the arrow cross the cell membrane, the fluorescent peaks are at a maximum, and through the center of the cell, the level of fluorescence decreases.
+
-
<br> <br>
+
-
 
+
-
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==
+
-
 
+
-
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/>
+
-
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|500px|middle]]:[[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]
+
-
</center>
+
-
 
+
-
 
+
 +
== '''Superfolder GFP-magnetosome gene protein fusions'''==
 +
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.''
 +
===sfGFP-MamK: Scaffold formation===
 +
<br>
 +
<center> [[File:Washington igem11 MamK fusion full 01.jpg|400px|middle]][[File:Washington igem11 MamK fusion gfp 01.jpg||400px|middle]]</center>
 +
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.
 +
Here is a video showing the filaments connecting the growing ''E. coli'':
 +
<html><center><iframe width="420" height="315" src="http://www.youtube.com/embed/BLLyUHrrcV4" frameborder="0" allowfullscreen></iframe></center></html>
 +
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. 
 +
 +
<gallery widths=180px heights=150px caption="More images of E.coli with sfGFP mamK fusion" >
 +
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) crop.jpg
 +
File:Washington igem11 SfGFP-K-1A3-100iptg-02(20ms exp) gfp.jpg
 +
File:Washington SfGFP K 4C5-col1 03 crop.jpg
 +
File:Washington igem11 SfGFP K 4C5-col1 03 gfp.jpg
 +
File:Washington igem11 SfGFP K 4C5-col1 05 crop.jpg
 +
File:Washington igem11 SfGFP K 4C5-col1 05 gfp.jpg
 +
File:Washington igem11 SfGFP K 4C5-col1 04 crop.jpg
 +
File:Washington igem11 SfGFP K 4C5-col1 04 gfp.jpg
 +
</gallery>
 +
<br>
 +
===sfGFP-MamI: Membrane localization===
 +
<center>[[File:Washington igem11 MamIfusion full.jpg|200px]][[File:Washington_igem11_MamIfusion_GFP.jpg|200px]][[File:Washington_igem11_mamI_graph.png|350px]] </center>
 +
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>
 +
-----
 +
=='''Construction of the R5 region of the Magnetosome Island in ''E.coli'' '''==
 +
After verifying that the construction of the sfGFP-MamK scaffold worked as expected, we proceeded to create a full assembly of the <i>mamAB</i> operon by building three super-assemblies: ''mamHIEJKL'', ''mamMNOPA'', and ''mamQRBSTUV''. The PCR products of these intermediate assemblies are shown below. The ''mamHIEJKL'' and ''mamQRBSTUV'' have been partially sequence-confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when cells with the ''mamHIEJKL'' construct were imaged, they appeared to be forming chains.<br/>
 +
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|500px|middle]]:[[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]
 +
</center>
 +
<br/><br/>
-
 
+
== '''A set of the 18 genes from the mamAB operon essential for magnetosome formation'''==
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
== '''A set of the 18 essential genes for the various steps of magnetosome formation'''==
+
Before piecing together the 16 kb genome of the mamAB gene cluster within the magnetosome island (MAI), we extracted out the genes in the following groups:  
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:  
-
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]
+
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel extracts of magnetosome gene clusters]]
{| class="wikitable"
{| class="wikitable"
|-
|-
Line 91: Line 100:
| 1002
| 1002
|-
|-
-
  |}.  
+
  |}.
-
+
== '''A table of individual gene functions ''' ==
 +
Please see our <i>mamAB</i> genes description [https://2011.igem.org/Team:Washington/Magnetosomes/mamDescriptions page].
 +
== ''' A table of Translational Efficiency''' ==
 +
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.
 +
{| class="wikitable" |left
 +
|-
 +
! Gene
 +
! AMB-1
 +
! E.coli
 +
! Change in %
 +
! Actual sequence inputted in the calculator (first 9nt and last 9nt)
 +
|-
 +
| mamH
 +
| 10821.63
 +
|10821.63
 +
| 0%
 +
| TCGGAGGTG......CCAGCACAA
 +
|-
 +
| mamI
 +
| 88.93
 +
| 88.93
 +
| 0%
 +
| TCGCTCTGC.....ATTGCTGGG
 +
|-
 +
| mamE
 +
| 37.82
 +
| 37.82
 +
| 0%
 +
| ATGGCCATG.....CTATCTGAT
 +
|-
 +
| mamJ
 +
| 105.9
 +
| 105.9
 +
| 0%
 +
| ACCGCAATG.....CCAGGGTGA
 +
|-
 +
| mamK
 +
| 1056.92
 +
| 1056.92
 +
| 0%
 +
| GTCATTTAG.....TGGCATCGA
 +
|-
 +
| mamL
 +
| 584.21
 +
| 584.21
 +
| 0%
 +
| CGGGGATAC.....CGTGCTGTT
 +
|-
 +
| mamM
 +
| 930.1
 +
| 930.1
 +
| 0%
 +
| GTCGGGGCT.....CGGCCTGGC
 +
|-
 +
| mamN
 +
| 413.73
 +
| 230.48
 +
| -44.29%
 +
| GAAGTCATG.....CGCCGTTAT
 +
|- 
 +
| mamO
 +
| 138.73
 +
| 138.73
 +
| 0%
 +
| TCCGAATGA.....CGTGTTTTG
 +
|-
 +
| mamP
 +
| 198.85
 +
| 198.85
 +
| 0%
 +
| TGATGATCA.....TGTTCTGGC
 +
|-
 +
| mamA
 +
| 14.29
 +
| 14.29
 +
| 0%
 +
| TAAAGTGAT.....CGAGGTCAC
 +
|-
 +
| mamQ
 +
| 22.41
 +
| 12.49
 +
| -44.27%
 +
| CCTTGCCGC.....TCCTTCATA
 +
|-
 +
| mamR
 +
| 8.04
 +
| 8.04
 +
| 0%
 +
| TCTCAACCA.....CGTGCAGGG
 +
|-
 +
| mamB
 +
| 16.56
 +
| 16.56
 +
| 0%
 +
| TCCAATCTT.....TGGGCCTTT
 +
|-
 +
| mamS
 +
| 32.86
 +
| 25.09
 +
| -23.65%
 +
| CACGGGCCT.....GATGGCCGA
 +
|-
 +
| mamT
 +
| 81.16
 +
| 81.16
 +
| 0%
 +
| TGGTGCAGT.....CTTGGGGAT
 +
|-
 +
| mamU
 +
| 33.58
 +
| 33.58
 +
| 0%
 +
| CGCTGACCA.....CGCCTGCCT
 +
|-
 +
| mamV
 +
| 1.61
 +
| 1.61
 +
| 0%
 +
| AATAAACCA.....TCGTGACCG
 +
|}.
-
== '''A table of individual gene functions ''' ==
+
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.
-
Please see the bottom of our [https://2011.igem.org/Team:Washington/Magnetosomes/Parts parts submitted page].
+
 
 +
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.
 +
 
 +
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.
 +
 
 +
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.

Latest revision as of 00:26, 29 October 2011


Magnetosome Toolkit: Results Summary


What’s in the Magnetosome Toolkit?

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



Superfolder GFP-magnetosome gene protein fusions

The two genes we characterized as fusions with superfolder GFP are mamK and mamI. They each perform core functions of magnetosome formation. MamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. MamI is a membrane-localized protein required for magnetosome vesicle formation that has also been shown to localize on the MamK filament. For more information, see the mamAB description page. Using our two genes of interest, we created C-terminal sfGFP fusions so we could track the localization of each gene separately within E.coli.

sfGFP-MamK: Scaffold formation


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

The results we obtained with our sfGFP fusions inside E. coli were comparable to those done through other studies in the host organism Magnetospirillum magneticum. Within AMB-1, MamK is a filament which runs along the long axis of the bacteria. In the our images of sfGFP-MamK, scaffold-like structures can be clearly seen running through the length of most of the cells, in some cases looping back within a single cell for "figure-8" shaped filaments. In other cases, the filaments seem to prevent the cells from dividing properly, resulting in long chains of E. coli. In our experimental result, there was an over- expression of mamK which connected the E.coli cells together.

Here is a video showing the filaments connecting the growing E. coli:

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.


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


sfGFP-MamI: Membrane localization

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

For mamI, the gene product localizes to the cell membrane, consistent with its known role in inner membrane vesicle invagination. 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.



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

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

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



A set of the 18 genes from the mamAB operon essential for magnetosome formation

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

Gel extracts of magnetosome gene clusters
Gene groups Length (bp)
mamHI 1541
mamE 2172
mamJ 1538
mamKL 1336
mamMN 2323
mamO 1914
mamPA 1493
mamQRB 2029
mamSTU 2030
mamV 1002
.

A table of individual gene functions

Please see our mamAB genes description page.

A table of Translational Efficiency

Using the RBS Calculator developed by the Salis, Mirsky, and Voigt (Link), we were able to estimate the relative translation rate of each gene from the mamAB operon.

Gene AMB-1 E.coli Change in % Actual sequence inputted in the calculator (first 9nt and last 9nt)
mamH 10821.63 10821.63 0% TCGGAGGTG......CCAGCACAA
mamI 88.93 88.93 0% TCGCTCTGC.....ATTGCTGGG
mamE 37.82 37.82 0% ATGGCCATG.....CTATCTGAT
mamJ 105.9 105.9 0% ACCGCAATG.....CCAGGGTGA
mamK 1056.92 1056.92 0% GTCATTTAG.....TGGCATCGA
mamL 584.21 584.21 0% CGGGGATAC.....CGTGCTGTT
mamM 930.1 930.1 0% GTCGGGGCT.....CGGCCTGGC
mamN 413.73 230.48 -44.29% GAAGTCATG.....CGCCGTTAT
mamO 138.73 138.73 0% TCCGAATGA.....CGTGTTTTG
mamP 198.85 198.85 0% TGATGATCA.....TGTTCTGGC
mamA 14.29 14.29 0% TAAAGTGAT.....CGAGGTCAC
mamQ 22.41 12.49 -44.27% CCTTGCCGC.....TCCTTCATA
mamR 8.04 8.04 0% TCTCAACCA.....CGTGCAGGG
mamB 16.56 16.56 0% TCCAATCTT.....TGGGCCTTT
mamS 32.86 25.09 -23.65% CACGGGCCT.....GATGGCCGA
mamT 81.16 81.16 0% TGGTGCAGT.....CTTGGGGAT
mamU 33.58 33.58 0% CGCTGACCA.....CGCCTGCCT
mamV 1.61 1.61 0% AATAAACCA.....TCGTGACCG
.

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.

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.

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.

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.

Retrieved from "http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results"