Difference between revisions of "Part:BBa K1189021"
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<h1>Fusion of ferrtin subunits linked to a DNA detector</h1> | <h1>Fusion of ferrtin subunits linked to a DNA detector</h1> | ||
| − | <p>Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 <a | + | <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 (see Figure 1). 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 <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189022">DNA sensing TALE (BBa_K1189022)</a> as part of their project, the <a href="http://2013.igem.org/Team:Calgary/Project/OurSensor">FerriTALE</a>. The TALE sequence was based off of <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K782004">BBa_K782004</a> from the <a href="http://2012.igem.org/Team:Slovenia">2012 Slovenia iGEM</a> team. The Calgary team used this TALE as a proof of concept for their DNA detector.</p> |
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| − | href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189024">light (BBa_K1189024) | + | |
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| − | </a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189025">heavy | + | |
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| − | (BBa_K1189025)</a> subunits (see Figure 1). It is ubiquitous across eukaryotic and | + | |
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| − | prokaryotic systems and is used to sequester intracellular iron (Chasteen <i>et al.</i>, | + | |
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| − | 1991). The <a href="http://2013.igem.org/Team:Calgary">2013 iGEM Calgary</a> used | + | |
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| − | ferritin’s iron core as a reporter and its protein shell to scaffold a <a | + | |
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| − | href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189022">DNA sensing TALE | + | |
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| − | (BBa_K1189022)</a> as part of their project, the <a | + | |
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| − | href="http://2013.igem.org/Team:Calgary/Project/OurSensor">FerriTALE</a>. The TALE sequence | + | |
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| − | was based off of <a href="https://parts.igem.org/wiki/index.php? | + | |
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| − | title=Part:BBa_K782004">BBa_K782004</a> from the <a | + | |
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| − | href="http://2012.igem.org/Team:Slovenia">2012 Slovenia iGEM</a> team. The Calgary team | + | |
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| − | used this TALE as a proof of concept for their DNA detector.</p> | + | |
<figure> | <figure> | ||
| − | <img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" | + | <img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300"> |
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| − | alt="Ferritin" width="300" height="300"> | + | |
<figcaption> | <figcaption> | ||
<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p> | <p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p> | ||
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<h1>Design features</h1> | <h1>Design features</h1> | ||
| − | <p><a href="https://parts.igem.org/Part:BBa_K1189021">BBa_K1189021</a> is a fusion of heavy | + | <p><a href="https://parts.igem.org/Part:BBa_K1189021">BBa_K1189021</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 the fused TALE in the 3D sphere surrounding ferritin. Additionally, di-subunits mandate a 1:1 ratio of heavy and light subunits, ensuring consistency in ferritin’s iron uptake dynamics. Moreover, these di-subunit fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (Dehal <i>et al.</i>, 2010).</p> |
| − | + | <p><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189022">TALE A (BBa_K1189022)</a> is attached to the N-terminus of the ferritin di-subunit. Given that the C-terminus of ferritin is oriented toward the core of ferritin, and that we wanted proteins to be displayed on the outside of the ferritin sphere, we selected the N-terminus (CITE ME). TALE A is connected to ferritin by a flexible <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189026">GS linker (BBa_1189022)</a>. The iGEM Calgary team made this decision because <a href="XXX">spatial modelling</a> suggested that TALEs require freedom of movement to allow for DNA binding. See Figure two for a diagram of BBa_K1189021.</p> | |
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| − | <p><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189022">TALE A | + | |
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| − | (BBa_K1189022)</a> is attached to the N-terminus of the ferritin di-subunit. Given that the | + | |
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| − | C-terminus of ferritin is oriented toward the core of ferritin, and that we wanted proteins | + | |
| − | + | ||
| − | to be displayed on the outside of the ferritin sphere, we selected the N-terminus (CITE | + | |
| − | + | ||
| − | ME). TALE A is connected to ferritin by a flexible <a | + | |
| − | + | ||
| − | href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189026">GS linker | + | |
| − | + | ||
| − | (BBa_1189022)</a>. The iGEM Calgary team made this decision because <a href="XXX">spatial | + | |
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| − | modelling</a> suggested that TALEs require freedom of movement to allow for DNA binding. | + | |
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| − | See Figure two for a diagram of BBa_K1189021.</p> | + | |
<figure> | <figure> | ||
| − | <img src="https://static.igem.org/mediawiki/2013/thumb/8/8d/BBa_1189021_SBOL.png/800px- | + | <img src="https://static.igem.org/mediawiki/2013/thumb/8/8d/BBa_1189021_SBOL.png/800px-BBa_1189021_SBOL.png" alt="TALEA-GS_linker_Ferritin" width="600" height="105"> |
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| − | BBa_1189021_SBOL.png" alt="TALEA-GS_linker_Ferritin" width="600" height="105"> | + | |
<figcaption> | <figcaption> | ||
| − | <p><b>Figure 2.</b> TALE A is directly fused via a GS linker to a di-hybrid | + | <p><b>Figure 2.</b> TALE A is directly fused via a GS linker to a di-hybrid ferritin fusion. This construct will scaffoled 12 TALE A to the outside of ferritin.</p> |
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| − | ferritin fusion. This construct will scaffoled 12 TALE A to the outside of ferritin.</p> | + | |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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<h3>Performance of BBa_K1189021 as a reporter</h3> | <h3>Performance of BBa_K1189021 as a reporter</h3> | ||
| − | <p>BBa_K1189021 was expressed in <i>E. coil BL21</i> and purified using metal affinity | + | <p>BBa_K1189021 was expressed in <i>E. coil BL21</i> and purified using metal affinity chromatography on an FPLC. The purified protein was chemically modified into <a href |
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| − | chromatography on an FPLC. The purified protein was chemically modified into <a href | + | |
<h1>References</h1> | <h1>References</h1> | ||
| − | <li>Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. | + | <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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| − | (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. | + | |
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| − | Analytical chemistry, 83(15), 5834-5843.</li> | + | |
<br></br> | <br></br> | ||
Revision as of 17:11, 1 November 2013
Fusion of ferrtin subunits linked to a DNA detector
Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 light (BBa_K1189024) and heavy (BBa_K1189025) subunits (see Figure 1). 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 a DNA sensing TALE (BBa_K1189022) as part of their project, the FerriTALE. The TALE sequence was based off of BBa_K782004 from the 2012 Slovenia iGEM team. The Calgary team used this TALE as a proof of concept for their DNA detector.
Figure 1. Ribbon visualization of a fully assembled ferritin protein.
Design features
BBa_K1189021 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 the fused TALE in the 3D sphere surrounding ferritin. Additionally, di-subunits mandate a 1:1 ratio of heavy and light subunits, ensuring consistency in ferritin’s iron uptake dynamics. Moreover, these di-subunit fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (Dehal et al., 2010).
TALE A (BBa_K1189022) is attached to the N-terminus of the ferritin di-subunit. Given that the C-terminus of ferritin is oriented toward the core of ferritin, and that we wanted proteins to be displayed on the outside of the ferritin sphere, we selected the N-terminus (CITE ME). TALE A is connected to ferritin by a flexible GS linker (BBa_1189022). The iGEM Calgary team made this decision because spatial modelling suggested that TALEs require freedom of movement to allow for DNA binding. See Figure two for a diagram of BBa_K1189021.
Figure 2. TALE A is directly fused via a GS linker to a di-hybrid ferritin fusion. This construct will scaffoled 12 TALE A to the outside of ferritin.
Results
Performance of BBa_K1189021 as a reporter
BBa_K1189021 was expressed in E. coil BL21 and purified using metal affinity chromatography on an FPLC. The purified protein was chemically modified into References
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2654
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3656