Our Platform

Our team set out to develop a nucleic acid biosensor to monitor Enterohemorhagic E. coli (EHEC) in beef cattle. Though we tailored our final system to the beef industry, the molecular components forming the FerriTALE have a myriad of other applications. These include detector TALEs which can be readily designed to other DNA targets, a ferritin protein shelled nanoparticle with interchangeable cores that impart novel function, and an interchangeable coiled-coil linker system to build intricate protein devices using ferritin as a scaffold. Together, these components form a customizable DNA biosensor, with individual components that could have diverse applications in future iGEM projects and beyond.

Detector

We selected Transcription Activator Like Effectors (TALEs) to bind and detect DNA in our system. These proteins were chosen because their DNA binding domains can be engineered to bind virtually any DNA sequence (Cermak et al., 2011). This means that we have built a DNA biosensor platform, where our system can be repurposed to detection of organisms in other applications by designing TALEs to different gene sequences. By combining alternative TALE proteins into our capture strip assay, our system could be relevant to other food industries, health care, bedside diagnostics, and biosecurity (Figure 1).

To enable the iGEM community to harness the FerriTALE to detect other DNA sequences, we added KasI restriction cut sites to the 3’ of detector Biobricks, allowing different TALEs to be introduced in the system (BBa_K1189029, BBa_K1189030, BBa_K1189032, and BBa_K1189033). This cut site enables traditional restriction enzyme cloning methods to combine FerriTALE proteins with different detector TALEs (Figure 2). Additionally, we improved two TALEs (BBa_K1189022, BBa_K1189023) previously submitted to iGEM to eliminate eukaryotic Kozak translation initiation sequences which prevent expression in prokaryotic systems. This modification serves as a template for how future teams should design TALEs so that they can use E. coli to manufacture custom FerriTALEs.

Ferritin— A protein shelled nanoparticle

Ferritin is an iron sequestering protein shelled nanoparticle which could benefit other teams as a platform system in how the iron core can be converted to other compounds with different functions. We demonstrated this in our own system where we chemically modified the iron core to make ferritin a robust colourmetric reporter. Other intriguing applications include making ferritin’s iron core magnetically active as magnetoferritin (Jordan et al. 2013), using ferritin as a nanocage for other metals, or the incorporation of other reporters such as quantum dots (Naito et al. 2013) (Figure 3). The applications are diverse when one considers how these different cores can be combined with other proteins by expressing ferritin shell subunits as protein fusions. Check out how we combined detector TALEs with Prussian blue ferritin using coiled-coil linkers.

The ferritin scaffold and coiled-coil linkers

Apart from its core, the ferritin nanoparticle is useful for iGEM teams as a self-assembling and spherical protein scaffold. Each of the 24 subunits forming ferritin can be fused proteins of interest, such that when the nanoparticle assembles, proteins surround the ferritin sphere (Kim et al., 2011). We used this in the FerriTALE to allow variation of the number of detector TALEs per ferritin reporter, enabling us to modulate sensitivity of the system. To improve flexibility of the type and composition of proteins which can be attached to ferritin, we introduced a system of complementary coiled-coils onto ferritin (BBa_K1189012, BBa_K1189013, BBa_K1189018). This makes our ferritin a platform since other teams can manufacture intricate protein nanoparticles by fusing their own proteins of interest to our coils (Figure 4). We are excited by how future teams might customize ferritin by interchanging different proteins and enzymes from the Parts Registry.

Additionally, these coils dimerize with high affinity and specificity and can be repurposed for affinity purification, capture systems, and development of self-assembling biomaterials (Apostolovic and Klok, 2008).