Part:BBa_K1189021

Fusion of ferrtin subunits linked to DNA detectors

Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 light (BBa_K1189024) and heavy (BBa_K1189025) subunits. 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 DNA sensing TALEs as part of their project, the FerriTALE (see Figure 1). TALE A was integated into the project as a proof of concept for the DNA detector. The TALE sequence was based off of BBa_K782004 from the 2012 Slovenia iGEM team.

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

BBa_K1189037 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).

Figure 2. Sample diagram of a TALE protein. Twelve proteins of this sort were fused to the exterior of ferritin nanoparticles with BBa_K1189021.

Design features

This part is a ferritin di-subunit that has an N-terminal fusion to a DNA detecting protein, TALEA (BBa_K1189022). These components are connected to ferritin by a flexible GS linker (BBa_1189022) to ameliorate concerns about how steric hindrance could inhibit performance of the TALE. This idea was based upon the work of Kim et al. (2011) who found that output from GFP fused to ferritin was improved in the presence of a linker.

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.

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