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TALEA-dife

Part:BBa_K1189021

Designed by: Chris Wintersinger, Denny Hoang, Taylor Remondini   Group: iGEM13_Calgary   (2013-09-17)
Revision as of 17:11, 1 November 2013 by Cmwinter (Talk | contribs)

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.

Ferritin

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.

TALEA-GS_linker_Ferritin

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

  • 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
      INCOMPATIBLE WITH RFC[21]
      Illegal BamHI site found at 2654
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 3656


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