Coding

Part:BBa_K1189018

Designed by: Chris Wintersinger, Denny Hoang, Taylor Remondini   Group: iGEM13_Calgary   (2013-09-17)
Revision as of 02:03, 1 November 2013 by Cmwinter (Talk | contribs)


A fusion of two ferrtin subunits

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 et al., 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).

<figure> <img src="463px-Assembled_FerriTALE.png" alt="BBa_K1189037 joined with DNA sensing TALEs" width="400" height="500"> <figcaption>

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.

</figcaption> </figure>

<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 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.

<figure> <img src="800px-BBa_1189018_SBOL.png" alt="BBa_K1189018 SBOL part figure" width="500" height="100"> <figcaption>

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

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<figure> <img src="UCalgary2013TRCoilflexibility.png" alt="FerriTALE Scaffold Modularity" width="800" height="219" > <figcaption>

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.</a>

</figcaption> </figure>

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.

Results

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>.


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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.

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    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
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 1289


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    Categories
    Parameters
    None