Linker

Introduction

In order to design a functional sensor, we needed to be able to link the detector to the reporter. We considered a direct linkage between the reporter and the detector, which we decided to pursue for the beta-lactamase as a reporter. We have also built and submitted a construct with the a direct linkage between the TALEs and the ferritin. However, due to the massive size of the finished product (~1300 kDa) we needed another system, a system which allows in vitro assembly of the reporter and the detector. This is why we looked into the use of E and K coiled-coils in our system. The addition of the coiled-coils gives us flexibility in our system to disassembly of the target-specific detector from the reporter in order to switch in another detector for a different application. In other words, the coiled-coils add re-usability to the system making it a great platform technology. In addition, ferritin serves as a great scaffolding protein, allowing us to stick multiple different detectors on it. We can also scale up the reporter response by adding reporters on ferritin, depending on the requirements of the system where the sensor is applied. Therefore, the ability to scale up and down allows us to incorporate informed design in our platform technology application.

What are E/K Coils?

E/K coils are synthetic coiled-coil domains designed to bind specifically to each other with high affinity and specificity (Litowski and Hodges, 2002) (Figure 1). They are composed of a heptad repeat of amino acids that form a coil structure that is able to interact with each other. These coils are able to interact with each other in an anti-parallel fashion, making them useful for applications such as peptide capture, protein purification and biosensors. For our project we decided to make use of the IAAL E3/K3 coils (BBa_K1189010, BBa_K1189011) due to the balance they offer between affinity and specificity (Table 1, Figure 2). These parts were already in the registry, however the DNA was never received, so we built, sequenced and re-submitted them.

How do These Coils Work?

These E3/K3 coils are able to form heterodimers due to the hydrophobic residues contained within the heptad repeat. In our case these heptad repeats are composed of isoleucine and leucine residues. These hydrophobic residues form the core of the binding domain of the coils, indicated in Figure 3 by the empty arrows. In order to prevent the homodimerization of these coils, charged residues are included in the design. For example, the electrostatic interactions between glutamic acid and lysine residues prevent an E coil from binding with an E coil. We selected the use of E3/K3 coiled-coils over other synthetic E/K coils, as the isoleucine residue present shows a significant increase in the heterodimerization over valine found in other coils. The alpha-helical propensity of the residues outside of the core interacting residues is also increased by utilizing an alanine residue instead of the serine residue found in ISAL and VSAL E/K coils. This selection maximizes the stability and specificity of the coils used in our system.

What did we construct?

The first construct we created involved attaching the previously mentioned E coil biobrick to the fusion of heavy and light chains of ferritin. (BBa_K1189018, BBa_K1189037) (Figure 4). This construct would assemble to form a 12-subunit nanoparticle with E coils on the outside that would each bind a protein containing the respective K coil. This means we can simply attach the K coil to a protein of choice, such as a TALE in order to bind it to the ferritin scaffold (BBa_K1189018, BBa_K1189037) Therefore we constructed the respective K coil-TALE fusion constructs, BBa_K1189029 and BBa_K1189030 (Figure 5 & 6). The ideal result of this system is that BBa_K1189029 and BBa_K1189030 will be bound to ferritin, resulting in a protein complex containing 12 TALE proteins bound to the ferritin scaffold through E/K coils. This system then allows the proteins to properly fold while at the same time allowing the ferritin nanoparticle to assemble without interference.

So do These Coils Actually Bind?

After putting in gratuitous effort to build parts containing these coils and successfully purifying these proteins we wanted to determine if the E and K coils interacted with each other. In order to characterize coil-coil interaction we performed an immunoprecipitation (IP) assay. We built a GFP with an E coil ( BBa_K1189014) and we also built TALE-B with a K coil (BBa_K1189030). To characterize the binding of the coils we pulled down with either an immunoglobulin G antibody (IgG) that serves as a negative control or with GFP antibody. The idea behind this experiment is to pull down the E coil which is fused to GFP with a GFP antibody and then probe with a anti-his antibody which recognizes the his tag on the TALE fused to the K coil. Upon interaction between the E and K coils we will see an output at approximately 86 kDa when probed with a His-antibody as seen in Figure 7. Our test groups included the coils by themselves and both the E and K coils put together in solution. As seen in Figure 7 a band appears only when we pull E and K coil with a GFP and probe with an anti-his antibody indicating the presence of both GFP and TALE in the elution solution indicating that the coils interact with each other.

Ferritin Scaffold

The ferritin scaffold we chose to use, BBa_K1189018, allows us to modulate our system as we wish. We determined the ferritin-coiled-coil system could be used for more than the detection of our E.coli sequence. The ferritin scaffold can detect other sequences of DNA by exploiting the ability of the TALE system to be easily engineered and modified, along with the interchangeability of the coiled-coil system. Thus by attaching K coils to TALEs that target alternative sequences, we can use this system as a platform for any variety of DNA sequences. In our system, we utilized two TALE proteins, TALE A and TALE B (BBa_K1189029, BBa_K1189030). These two parts were both created with the K coil, allowing us to use the same ferritin scaffold (BBa_K1189018, BBa_K1189037) to anchor our proteins. This proved to be incredibly useful in binding the TALE proteins to nitrocellulose, a process that typically requires chemical modifications and specialized proteins in order to ensure the active site of these proteins are functional. Instead, we could interchange TALE A for TALE B on the ferritin scaffold due to our coiled-coil system. The protein complex can then simply be blotted onto our nitrocellulose strip, ensuring at minimum, the upwards-facing TALEs will be able to bind DNA. Furthermore, we can expand this system towards proteins and other molecules as well by replacing the TALEs with antibodies or other binding proteins. The use of coils on the ferritin scaffold also proves useful to prevent the TALE proteins in our FerriTALE from interrupting the self-assembly of ferritin due to our large size. An experiment which can be viewed on our Prussian blue ferritin page was used to demonstrate the viability of these coils for bringing our FerriTALE together. By examining the catalytic activity of our Prussian blue ferritin we could evaluate whether the direct fusion of TALEs or the use of coils was more effective for our system. The results of this experiment showed that using coils to bind our TALEs is the most effective route.

Another aspect of the ferritin nanoparticle that can be exploited is its composition, being composed of 24 subunits, as well as its potential as a novel reporter. Ferritin is typically composed of two chains, heavy and light. In our system, each one of these subunits can be bound to a reporter, effectively amplifying our catalytic activity by 24 due to the increased number of bound reporter proteins. But this activity can be scaled down through previously reported heavy-light chain fusions (Huh and Kim, 2003). Rather than 24 subunits the nanoparticle would be reduced to 12 subunits, as in our system, and thus the number of reporter proteins would decrease accordingly. We can scale this down even further due to our ability to transform the iron core of the protein into a catalytic active substance called Prussian Blue (Zhang et al., 2013). Thus, instead of multiple bound reporter proteins we can further scale down the output of our system to one with essentially one reporter. The ability of our system to scale up and down according to the need of detection means this system is applicable in areas ranging from low sensitivity to high sensitivity.