Difference between revisions of "Part:BBa K1189020"
 
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<p>This light ferritin chain comes from humans. This part along with heavy ferritin (<partinfo>BBa_K1189025</partinfo>), form the ferritin nanoparticle, an iron-storage particle made up of 24 subunits. 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>Light ferritin subunit fused to an E coil</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. 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_B0010</partinfo>, & <partinfo>BBa_B0012</partinfo>) and a his-tag in order for us to induce protein expression as well as purify it.
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<p>BBa_K1189020 is the light 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">BBa_R0010</a>) and a strong RBS (<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>). Twenty-four of these light 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 light ferritin purportedly contributes to nucleation to initiate iron core formation in ferritin molecules (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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<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_K1189020 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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<img src="https://static.igem.org/mediawiki/2013/thumb/4/43/BBa_K1189020_SBOL.png/800px-BBa_K1189020_SBOL.png" alt="BBa_K1189019 SBOL part figure" width="500" height="100">
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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 light 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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<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 light ferritin chain was inspired by human light ferritin (<a href="http://www.uniprot.org/uniprot/P02792">P02792 [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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<br></br>
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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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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 07:14, 2 November 2013

Light ferritin subunit fused to an E coil

BBa_K1189020 is the light subunit of human ferritin fused to a linker E coil (BBa_K1189011), under control of a lactose inducible promoter (BBa_R0010) and a strong RBS (BBa_B0034). Twenty-four of these light 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 light ferritin purportedly contributes to nucleation to initiate iron core formation in ferritin molecules (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_K1189020 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 light 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 light ferritin chain was inspired by human light ferritin (P02792 [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 881
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 746