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. This assay indicated successful purification. We also viewed the encapsulin under a fluorescent microscope with inconclusive results. The protein concentration was evaluated with a NanoDrop.

Figure 2: SDS Page Results

Figure 3: Fluorescence Microscopy (possible encapsulin clusters circled in white)

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.