Difference between revisions of "Part:BBa K1189018:Experience"
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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<h1>A fusion of two ferrtin subunits</h1>
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how you used this part and how it worked out.
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===Applications of BBa_K1189018===
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<p>Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189024">light (BBa_K1189024)</a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189025">heavy (BBa_K1189025)</a> subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron (Chasteen <i>et al.</i>, 1991). The <a href="http://2013.igem.org/Team:Calgary">2013 iGEM Calgary</a> used ferritin’s iron core as a reporter and its protein shell to scaffold <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor/Detector">DNA sensing TALEs</a> as part of their project, the <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor">FerriTALE</a> (see Figure 1).</p>
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<p>We performed a kinetic analysis of our Prussian blue ferritin. We included a comparison of Prussian blue horse spleen ferritin to regular horse spleen ferritin for both TMB and ABTS (Figures 1, 2). For both of the substrates we can see that normal ferritin has a very low catalytic activity compared to our modified ferritin. Using this data were able to determine the Michaelis-Menten catalytic constants for Prussian blue ferritin with different substrates.</p>
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<figure>
 
<figure>
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<img src="https://static.igem.org/mediawiki/2013/3/36/UCalgary2013TRTmb6ulgraph.png" alt="Prussian Blue Ferritin and TMB" width="800" height="439">
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<img src="https://static.igem.org/mediawiki/2013/thumb/3/34/Assembled_FerriTALE.png/463px-Assembled_FerriTALE.png" alt="BBa_K1189037 joined with DNA sensing TALEs" width="400" height="500">
 
<figcaption>
 
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<p><b>Figure 1.</b> Measurements of the absorbance of the 650nm light by the substrate TMB over a period of 600 seconds. 6 µL of 10 mg/mL substrate was used in a 242 µL reaction volume.Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are TMB and hydrogen peroxide, and TMB only. Standard error of the mean bars are based on a sample size where n=8. Substrate and hydrogen peroxide sample data is not clearly visible as it is in line with the substrate only data. </p>
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<p><b>Figure 1.</b> 3D <i>in silico</i> rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. The iron core is chemically modified and use to show when TALEs are bound to DNA. The TALEs are one specific, particular application of the ferritin E coil di-subunit fusion. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.</p>
 
</figcaption>
 
</figcaption>
 
</figure>
 
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<figure>
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<p><a href="https://parts.igem.org/Part:BBa_K1189037">BBa_K1189037</a> is a fusion of heavy and light ferritin subunits, such that ferritin nanoparticles are formed from 12 di-subunits. The rationale for this design is that it reduces the number of N-termini on ferritin to which proteins can be fused by half, which is important for lessening potential steric hindrances among fused proteins in the 3D sphere surrounding ferritin. Additionally, di-subunits mandate a 1:1 ratio of heavy and light subunits which ensures consistency in ferritin’s ability to uptake iron. Moreover, these fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (Dehal <i>et al.</i>, 2010).</p>
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<img src="https://static.igem.org/mediawiki/2013/1/15/UCalgary2013TRABTS8ulgraph.png" alt="Prussian Blue Ferritin and ABTS" width="800" height="433">
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<figcaption>
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<p><b>Figure 2.</b> Measurements of the absorbance of the 415nm light by the substrate ABTS over a period of 600 seconds. 8 µL of 10 mg/mL substrate was used in a 242 µL reaction volume. Commercial Prussian blue ferritin ( 10 µL of 0.022 mg/mL sample) is represented by the blue data points. Orange data points are a negative control using standard ferritin (10 µL of 0.047 mg/mL sample). Negative controls are ABTS and hydrogen peroxide, and ABTS only. Standard error of the mean bars are based on a sample size where n=8.</p>
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</figcaption>
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</figure>
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<p>In order to complete our kinetic analysis we had to determine the catalytic properties of our Prussian blue ferritin according to the Michaelis-Menten kinetic model. For these tests we varied the colourimetric substrate concentrations (ABTS and TMB) (Figures 3,4). We also varied the hydrogen peroxide concentration in association with TMB as this the first chemical compound that will react in the system (Figure 5).</p>
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<h1>Design features</h1>
  
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<figure>
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<p>This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker (Figure 2). The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing user to build intricate nanoparticle devices with myriad proteins. See Figures 3 application examples.</p>
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<img src="https://static.igem.org/mediawiki/2013/2/20/UCalgary2013TRPBFABTSmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with ABTS" width="800" height="436">
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<figcaption>
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<p><b>Figure 3.</b> Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of ABTS. Absorbance readings were taken at 415 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 6).</p>
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</figcaption>
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</figure>
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<img src="https://static.igem.org/mediawiki/2013/c/c7/UCalgary2013TRPBFTMBmichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin with TMB" width="800" height="435">
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<img src="https://static.igem.org/mediawiki/2013/thumb/b/b4/BBa_1189018_SBOL.png/800px-BBa_1189018_SBOL.png" alt="BBa_K1189018 SBOL part figure" width="500" height="100">
 
<figcaption>
 
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<p><b>Figure 4.</b> Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of TMB. Absorbance readings were taken at 650 nm. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data (eg. Figure 5).</p>
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<p><b>Figure 2.</b> Ferritin di-subunit fused to an E coil. The E coil allows binding to other proteins expressing a complementary K coil.</p>
 
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<br></br>
  
 
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<img src="https://static.igem.org/mediawiki/2013/e/e5/UCalgary2013TRPBFTMBGHydrogenperoxidemichaelismentengraph.png" alt="Michaelis-Menten Plot for Prussian Blue Ferritin Based on Hydrogen Peroxide (with TMB)" width="800" height="434">
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<img src="https://static.igem.org/mediawiki/2013/0/07/UCalgary2013TRCoilflexibility.png" alt="FerriTALE Scaffold Modularity" width="800" height="219" >
 
<figcaption>
 
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<p><b>Figure 5.</b> Michaelis-Menten kinetic plot for commercial Prussian blue ferritin based on varying concentrations of hydrogen peroxide. Absorbance readings were taken at 650 nm which measure the breakdown of TMB. Velocities were generated from the average slope of eight data sets. Standard error of the mean bars are not displayed but are present in the foundational data.</p>
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<p><b>Figure 3.</b> Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.</a></p>
 
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</figure>
 
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<center><b>Table 1.</b> Catalytic constants for our Prussian blue ferritin</center>
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<p>This part is identical to <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_1189037</a>, except this part has no his purification tag.</p>
  
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<center><table width="800" border="1">
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<h1>Results</h1>
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  <tr>
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    <td>Catalyst</td>
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    <td>Enzyme Concentration (M)</td>
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    <td>Substrate</td>
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    <td>K<sub>m</sub> (mM)</td>
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    <td>V<sub>max</sub> (Ms<sup>-1</sup>)</td>
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    <td>K<sub>cat</sub> (s<sup>-1</sup>)</td>
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    <td>K<sub>cat</sub>/K<sub>m</sub> (M<sup>-1</sup>s<sup>-1</sup>)</td>
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  </tr>
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  <tr>
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    <td>Prussian Blue Ferritin</td>
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    <td>1.31 x 10<sup>-9</sup></td>
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    <td>ABTS</td>
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    <td>0.448</td>
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    <td>1.25 x 10<sup>-8</sup></td>
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    <td>9.51</td>
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    <td>2.12 x 10<sup>4</sup></td>
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  </tr>
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    <td>Prussian Blue Ferritin</td>
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    <td>1.31 x 10<sup>-9</sup></td>
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    <td>TMB</td>
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    <td>0.0432</td>
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    <td>1.12 x 10<sup>-7</sup></td>
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    <td>85.3</td>
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    <td>1.97 x 10<sup>6</sup></td>
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  </tr>
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    <td>Prussian Blue Ferritin</td>
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    <td>1.31 x 10<sup>-9</sup></td>
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    <td>H<sub>2</sub>O<sub>2 </sub> (TMB)</td>
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    <td>0.0176</td>
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    <td>1.31 x 10<sup>-8</sup></td>
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    <td>11.1</td>
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    <td>6.28 x 10<sup>5</sup></td>
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  </tr>
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</table></center>
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<p>Please see the results from <a href="https://parts.igem.org/Part:BBa_K1189037#Results">BBa_K1189037</a>. The coding sequence is identical to BBa_K1189018, except for the his tag that we required to purify and characterize this part. Additionally, this page discusses how we converted this part into a reporter, <a href="https://parts.igem.org/Part:BBa_K1189037#Reporter data">Prussian blue ferritin</a>.</p>
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<br></br>
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<h1>References</h1>
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<li>Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.</li>
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<li>Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody‐like proteins. Biotechnology journal, 5(6), 596-604.</li>
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===User Reviews===
 
===User Reviews===

Latest revision as of 00:36, 1 November 2013

A fusion of two ferrtin subunits

Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 light (BBa_K1189024) and heavy (BBa_K1189025) subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron (Chasteen et al., 1991). The 2013 iGEM Calgary used ferritin’s iron core as a reporter and its protein shell to scaffold DNA sensing TALEs as part of their project, the FerriTALE (see Figure 1).

BBa_K1189037 joined with DNA sensing TALEs

Figure 1. 3D in silico rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. The iron core is chemically modified and use to show when TALEs are bound to DNA. The TALEs are one specific, particular application of the ferritin E coil di-subunit fusion. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.

BBa_K1189037 is a fusion of heavy and light ferritin subunits, such that ferritin nanoparticles are formed from 12 di-subunits. The rationale for this design is that it reduces the number of N-termini on ferritin to which proteins can be fused by half, which is important for lessening potential steric hindrances among fused proteins in the 3D sphere surrounding ferritin. Additionally, di-subunits mandate a 1:1 ratio of heavy and light subunits which ensures consistency in ferritin’s ability to uptake iron. Moreover, these fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (Dehal et al., 2010).

Design features

This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker (Figure 2). The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing user to build intricate nanoparticle devices with myriad proteins. See Figures 3 application examples.

BBa_K1189018 SBOL part figure

Figure 2. Ferritin di-subunit fused to an E coil. The E coil allows binding to other proteins expressing a complementary K coil.



FerriTALE Scaffold Modularity

Figure 3. Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.

This part is identical to BBa_1189037, except this part has no his purification tag.

Results

Please see the results from BBa_K1189037. The coding sequence is identical to BBa_K1189018, except for the his tag that we required to purify and characterize this part. Additionally, this page discusses how we converted this part into a reporter, Prussian blue ferritin.



References

  • Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.
  • Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody‐like proteins. Biotechnology journal, 5(6), 596-604.



    • User Reviews

    UNIQ8996d0119adf5736-partinfo-00000001-QINU UNIQ8996d0119adf5736-partinfo-00000002-QINU