Difference between revisions of "Part:BBa K1189019"
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<partinfo>BBa_K1189019 short</partinfo>
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<p>This heavy ferritin chain comes from humans. This part along with light ferritin (<partinfo>BBa_K1189024</partinfo>), form the ferritin nanoparticle, an iron-storage particle made up of 24 subunits (Lawson <i>et al.</i>, 1991). The formed nanoparticle is highly robust, remaining stable at extreme pHs and temperatures. The difference between light ferritin is that this chain contains a ferroxidase centre.
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<h1>Fusion of ferrtin subunits linked to a DNA detector</h1>
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This nanoparticle can also be used as a reporter when the iron core is modified with potassium ferrocyanide to form Prussian Blue (Zhang <i>et al.</i>, 2013). The Prussian Blue ferritin can then act as a peroxidase mimic, similar to horseradish peroxidase, resulting in colour changes in the presence of hydrogen peroxide, and TMB or ABTS.
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We added the lacI promoter (<partinfo>BBa_J04500</partinfo>),  double terminator (<partinfo>BBa_B0015</partinfo>) and a his-tag in order for us to induce protein expression as well as purify it.
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</p>
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===Applications of BBa_K1189019===
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This protein was successfully expressed and purified through the use of his-tags. WESTERN BLOT STUFF
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We then modified the iron core in order to convert the protein into a peroxidase-like catalyst. Initial colourimetric testing was conducted and it was shown to be effective as the catalyst. PICTURE Qualitative kinetics testing was also conducted for commercial horsespleen ferritin to determine pH and temperature optimums, as well as Michaelis-Menten kinetics for the TMB substrates. PICTURESSSSSs
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<p>BBa_K1189019 is the heavy subunit of human ferritin fused to a linker E coil (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189011">BBa_K1189011</a>), under control of a lactose inducible promoter (<a href="https://parts.igem.org/Part:BBa_R0010">R0010</a>) and a strong RBS (<a href="https://parts.igem.org/Part:BBa_B0034">B0034</a>). Twenty-four of these heavy subunits will assemble to form a protein shelled, iron sequestering nanoparticle (Chasteen <i>et al.</i>, 1999) (see Figure 1). Ferritin is ubiquitous across prokaryotic and eukaryotic systems and is used to buffer intracellular iron by crystallizing it in its core using (Chasteen <i>et al.</i>, 1999). The heavy chain contains ferroxidase protein domains oriented toward ferritin's to cause the oxidation of intracellular iron from Fe^2+ to Fe^3+ and initiate formation of a ferrihydrite core. (Chasteen <i>et al.</i>, 1999). These nanoparticles are robust, remain stable at extreme pHs, withstand temperature variations, and can be used as a protein scaffold (Kim <i>et al.</i>, 2011)</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300">
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<figcaption>
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<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
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<h1>Design features</h1>
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<p>BBa_K1189019 has an N-terminal fusion to an E coil connected to ferritin by a <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189026">GS linker</a> (Figure 2).</p>
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<figure>
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<img src="http://2013.igem.org/File:BBa_1189019_SBOL.png" alt="BBa_K1189019 SBOL part figure" width="500" height="100">
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<figcaption>
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<p><b>Figure 2.</b>  E coil fused via a <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189026">GS linker</a> to the heavy ferritin subunit under control of the LacI inducible promoter and a strong RBS. This construct will for a spherical nanoparticle which can bind up to 24 proteins of interest assuming they are expressed in fusion with the complementary K coil.</p>
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</figure>
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<p>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 users to build intricate nanoparticle devices with myriad proteins. See Figures 3 application examples.</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2013/0/07/UCalgary2013TRCoilflexibility.png" alt="FerriTALE Scaffold Modularity" width="800" height="219" >
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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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<p>This heavy ferritin chain was inpsired by human heavy ferritin (<a href="http://www.uniprot.org/uniprot/P02794">P02794 [UniParc]</a>), codon optimized for <i>E. coli K12</i>, and commercially synthesized as shown in Figure 2. The iGEM Calgary team switched this construct into pSB1C3.</p>
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<h1>Results</h1>
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<p>The 2013 iGEM Calgary team did not make use of this sequence per se. Rather, they replicated this sequence using PCR, and integrated it into other constructs for their final system (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189018">BBa_K1189018</a>, <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189021">BBa_K1189021</a>, and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_K1189037</a>). Please see these respective pages for characterization data of these respective systems.</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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<li>Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. Analytical chemistry, 83(15), 5834-5843.</li>
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<br></br>
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Revision as of 06:54, 2 November 2013

Fusion of ferrtin subunits linked to a DNA detector

BBa_K1189019 is the heavy subunit of human ferritin fused to a linker E coil (BBa_K1189011), under control of a lactose inducible promoter (R0010) and a strong RBS (B0034). Twenty-four of these heavy subunits will assemble to form a protein shelled, iron sequestering nanoparticle (Chasteen et al., 1999) (see Figure 1). Ferritin is ubiquitous across prokaryotic and eukaryotic systems and is used to buffer intracellular iron by crystallizing it in its core using (Chasteen et al., 1999). The heavy chain contains ferroxidase protein domains oriented toward ferritin's to cause the oxidation of intracellular iron from Fe^2+ to Fe^3+ and initiate formation of a ferrihydrite core. (Chasteen et al., 1999). These nanoparticles are robust, remain stable at extreme pHs, withstand temperature variations, and can be used as a protein scaffold (Kim et al., 2011)

Ferritin

Figure 1. Ribbon visualization of a fully assembled ferritin protein.

Design features

BBa_K1189019 has an N-terminal fusion to an E coil connected to ferritin by a GS linker (Figure 2).

BBa_K1189019 SBOL part figure

Figure 2. E coil fused via a GS linker to the heavy ferritin subunit under control of the LacI inducible promoter and a strong RBS. This construct will for a spherical nanoparticle which can bind up to 24 proteins of interest assuming they are expressed in fusion with the complementary K coil.

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 users to build intricate nanoparticle devices with myriad proteins. See Figures 3 application examples.

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 heavy ferritin chain was inpsired by human heavy ferritin (P02794 [UniParc]), codon optimized for E. coli K12, and commercially synthesized as shown in Figure 2. The iGEM Calgary team switched this construct into pSB1C3.

Results

The 2013 iGEM Calgary team did not make use of this sequence per se. Rather, they replicated this sequence using PCR, and integrated it into other constructs for their final system (BBa_K1189018, BBa_K1189021, and BBa_K1189037). Please see these respective pages for characterization data of these respective systems.



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.
  • Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. Analytical chemistry, 83(15), 5834-5843.



    • Sequence and Features


    Assembly Compatibility:
    • 10
      COMPATIBLE WITH RFC[10]
    • 12
      COMPATIBLE WITH RFC[12]
    • 21
      COMPATIBLE WITH RFC[21]
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 230
      Illegal AgeI site found at 905
    • 1000
      COMPATIBLE WITH RFC[1000]