Part:BBa_K1189037
Ferrtin fusions
Ferritin is an 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. The 2013 iGEM Calgary used ferritin’s iron core as a reporter and its protein shell to scaffold DNA sensing TALEs (see Figure 1).
Figure 1. 3D in silico rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. TALEs are used to sense DNA while the iron core is used to report its presence. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.
This particular version is a fusion of heavy and light ferritin subunits, such that 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 ensure 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 (cite).
Design features
This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker. 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 2 application examples.
FIGURE TWO
This part is identical to BBa_1189018, with the exception of a his-tag for purification.
Results
The 2013 iGEM Calgary successfully expressed and purified this protein in pSB1C3 and per this part sequence exactly using and FPLC and metal affinity purification of the his tag. Please see the experience page for data on another expression vector.
GEL
This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 4 shows that this part with coiled-coils performs better as a reporter than direct fusions to TALEs (BBa_K118021). It seems that large protein fusions reduce effectiveness of ferritin as a reporter. Figure 5 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.
Figure 4 and 5
Please see the experience page for a detailed analysis of how Prussian blue ferritin performs as a reporter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1307
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