Figure 1: Biology Engineering Cycle
PCR Optimization
While our PCR experiment to add an alpha factor sequence to the encapsulin was not a standout success, its optimization did follow the engineering cycle of designing, building, testing, and learning, and then repeating this process iteratively.
Design We first designed the primers for inverse fusion PCR, considering factors like melting temperature and linker position in designing the sequences.
Build We then used these primer sequences to run inverse fusion PCR.
Test We tested the results by running gel electrophoresis and sequencing the plasmids. While certain gels looked promising, successful sequencing proved elusive.
Learn When the results deviated from our expectations, these discrepancies were analyzed and parameters like primer concentration, annealing temperature, and polymerase type were adjusted before repeating the cycle.
Encapsulin Protein Purification
Likewise for our purification of the encapsulins without alpha factor, we also followed the principles of the engineering cycle.
Design We first designed a plasmid containing the encapsulin protein carrying an mNeonGreen fluorescent cargo for visualization and a his-tag used for protein purification.
Build Then, we transformed the plasmid into competent BL21 E. Coli cells for the protein to be expressed. Afterward, we performed encapsulin protein purification using the his-tag. In doing so, the encapsulin protein was “built” by bacterial machinery.
Test To evaluate our results, we ran an SDS page gel to confirm the size of the protein matched the expected size of the encapsulin. We also viewed the encapsulin under a fluorescent microscope. Both of these evaluations indicated successful protein purification. The protein concentration was evaluated with a NanoDrop.
Figure 2: SDS Page Results
Figure 3: Fluorescence Microscopy (encapsulin circled in white)
Targeted Delivery and Controlled Immune Responses
A key advantage with encapsulins is the ability to modify the outer shell of the encapsulin to contain a variety of cell receptors which can make it highly selective. This is a critical feature for drug delivery as it would increase the efficiency of the drug being delivered without excess. Furthermore, this reduces potential side effects and complications with the delivery as the cargo will have limited interaction with other proteins or cells besides the intended target. Experiments have already shown that encapsulins are a viable drug delivery platform specifically for biomolecules. This can be seen with the fusion of SP94, a hepatocellular carcinoma cell-targeting peptide, to the encapsulin (EncTm) [2]. The specific cargo that was encapsulated was aldoxorubicin, an acid-sensitive prodrug. The experiments showed similar cell viability between encapsulated doxorubicin and free doxorubicin.
In addition, the ability to add various proteins to the surface of encapsulins has lead to innovative ways to induce various immune responses from cells. This would prove useful as it would help researchers further understand mechanisms behind immune responses and simulate controlled immune responses in the lab utilizing modified encapsulins. In summary, the versatility of the encapsulin structure gives it the potential to act as various pseudoviruses in research. This occurs in both empty encapsulins and cargo-loaded encapsulins. This is seen specifically when an experiment shows a major glycoprotein, gp350, for the Epstein-Barr virus fused to an Encapsulin. This would induce a strong immune response in both mice and non-human primates [3].
![Design of Encapsulin-based Vaccine [Kanekiyo et al., 2015]](https://static.igem.org/mediawiki/2021/9/9e/T--Michigan--img--vaccine.jpg)
Figure 4: Design of Encapsulin-based Vaccine [Kanekiyo et al., 2015]
Learn While the assays seemed to indicate successful encapsulin purification, a low protein concentration led us to believe that higher specific activity could be achieved in further iterations of the engineering cycle.