Introduction

Throughout this project, our team has conducted several key tests, including gel electrophoresis, SDS-PAGE, BCA test (only in cycle 2), and ELISA, each serving an important role in analyzing and verifying our proteins. In the two cycles, we initially carried out gel electrophoresis to confirm the incorporation of the designed gene into the bacterial hosts. Upon obtaining successful results from gel electrophoresis, we proceeded with plasmid transformation into the bacteria to facilitate protein expression. The transformed bacteria were then grown through cell culture to express the target protein. After that, a BCA test was conducted to find out the initial concentration of our samples. Subsequently, SDS-PAGE was conducted to verify the expression of the target protein. Finally, ELISA was employed to evaluate the binding affinity of the fusion peptides. Below are the successful results from these experiments.

Cycle 1

In the first cycle, we designed a fusion protein utilizing PEP-1 and FWT, which consists of FRATtide, Wnt3α, and TP1. Additionally, we incorporated another fusion peptide known as BC, which consists of BDNF and CDR1. For the expression of our genes and proteins, we have used DH5-α and BL21 strains.

GEL ELECTROPHORESIS

We used gel electrophoresis for separating proteins, DNA, and RNA based on their size and charge. It operates on the principle of applying an electric field to move charged molecules through a gel matrix. Molecules separate based primarily on size and charge, producing distinctive bands. Successful results are indicated by clear, well-resolved bands that correspond to expected sizes, while smearing, distortion, or unexpected bands suggest experimental failure, degradation, or contamination.

To confirm that the bacteria contain our designed gene, we used gel electrophoresis to verify the presence of a band matching the expected size of our gene.

Fig 1 FWT Gel electrophoresis

Fig 2 BC Gel electrophoresis

Result

In the pictures above, we can see the FWT band positioned around 1500 bps, while the BC band is at around 1000 bps. Both of them match our estimated base pair lengths, leading to the conclusion that our design was functioning as intended.

Transformation and cell culture

Fig 3 Successful transformation of FWT, BC to DH5-α

Fig 4 Successful transformation of FWT, BC to BL21

Fig 5 Successful culture of FWT, BC

Result

In the pictures above, we have done transformation of BC, FWT to BL21 and DH5-α. After the transformation success, we have cultured the cells with our plasmid.

SDS-PAGE

SDS-PAGE is a technique commonly used in synthetic biology experiments to separate proteins based on their molecular weight. In this process, proteins are denatured and uniformly coated with negative charges, allowing for separation based purely on size. After denaturation, the proteins are loaded onto a polyacrylamide gel and subjected to an electric field. The proteins migrate through the gel, with smaller proteins moving faster than larger ones. The success of the technique is evaluated by the presence of sharp, distinct bands at the expected molecular weights. If issues arise, troubleshooting typically focuses on the shape of the bands, the resolution of the gel, or the integrity of the gel itself.

In our project, SDS-PAGE was used to confirm the presence of our targeted proteins by verifying their expected molecular weights on the gel.

Fig 6 SDS-PAGE results of FWT and BC

Result

In the first cycle, the results were successful and aligned with our expectations, showing a clear and linear result. Both the FWT and BC lanes matched our estimated kDa, which is approximately 35 kDa. This outcome verifies the presence of our protein, confirming that both transformations of FWT and BC are successful. With these results, we can proceed to the next steps of our testing, which include conducting an ELISA kit.

ELISA

ELISA kits quantify specific proteins or antigens by using antibody binding and enzymatic colorimetric reactions. The sample antigen binds to a capture antibody on a microplate, then a detection antibody linked to an enzyme is added. Substrate addition produces a color change proportional to antigen concentration. Successful assays produce clear, reproducible signals with controls ensuring specificity and sensitivity. Data are analyzed using standard curves to determine precise protein concentrations, with options for multiplexing or kinetic measurements for enhanced analysis.

Result

In our initial ELISA assay, a standard curve quality of an ideal R² value was obtained in the BC component, confirming the structural integrity and functional efficacy of our DNA construct. For the color intensity, the BC component exhibited a strong yellow signal, indicating substantial protein expression and providing a solid foundation for its further development. In contrast, the absence of a yellow signal and detectable absorbance for the FWT component suggests a lack of protein production.

Cycle 2

In the second cycle, we began by replacing FWT with FT while keeping BC unchanged. Additionally, we substituted BL21 cells with SHuffle cells to enhance protein expression. The tests performed in cycle 1, including SDS-PAGE, gel electrophoresis, and ELISA, were repeated to test our newly designed plasmid.

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Gel electrophoresis

Gel electrophoresis was conducted, and the results indicated the success of our DNA expression in bacterial cells with the newly designed plasmid.

Fig 7 BC and FT Gel electrophoresis

Result

Similar to cycle 1, we confirmed the presence of our targeted gene by conducting gel electrophoresis. The results of the experiment were consistent with our expected bps, which are around 1000 bps, confirming the presence of FT, BC, and other separated proteins’ DNA and indicating that both transformations for BC and FT were successful. This enables us to move forward with additional tests.

Transformation and cell culture

Fig 8 Successful transformation of FT, BC to DH5-α

Fig 9 Successful transformation of FT, BC to SHuffle cells

Fig 10 Successful culture of FT, BC

SDS-PAGE

SDS-PAGE was conducted to check the existence of our protein.

Fig 11 SDS-PAGE results of FT and BC

Result

In the second cycle, we verified the presence of our recombinant plasmid using SDS-PAGE analysis. The results aligned with our expectations, showing bands in all lanes at approximately 35 kDa—our predicted molecular weight—verifying the existence of our protein once again and ensuring that both transformations for BC and FT succeeded. Moreover, the protein expression levels in SHuffle cell cultures were higher than those in BL21, confirming our hypothesis. These successful outcomes enabled us to proceed with further tests.

BCA

The Bicinchoninic Acid (BCA) protein assay works by first reducing copper ions (Cu²⁺) to cuprous ions (Cu¹⁺) in an alkaline environment, a reaction facilitated by the peptide bonds and certain amino acid residues like cysteine, tyrosine, and tryptophan in the protein sample. The cuprous ions then react with bicinchoninic acid to form a purple-colored complex that strongly absorbs light at 562 nm. The intensity of this color change is directly proportional to the protein concentration in the sample. By measuring the absorbance of the purple complex using a spectrophotometer and comparing it to a standard curve made from known protein concentrations, the total protein content can be accurately quantified. The assay is typically incubated at 37 to 60 °C to enhance sensitivity and achieve consistent results.

Fig 12 standard curve of BCA

Fig 13 Results of BCA

Result

We have conducted a BCA test. The R² value of the standard curve is 0.996. The concentrations of our refolded protein has been discovered, which is 0.42121478 mg/mL

ELISA

After obtaining successful results with SDS-PAGE and gel electrophoresis, we proceeded to ELISA tests to evaluate the binding affinity of the fusion peptides. We have put the ELISA kit assays up for an absorbance test, the results are shown below.

Fig 14 Elisa results of ANP32A

Fig 15 ELISA graph of ANP32A

Fig 16 Elisa results of TrkB, PI3k, NOS, GSK3.

Fig 17 ELISA graph of TrkB.

Fig 18 ELISA graph of PI3K.

Fig 19 ELISA graph of NOS.

Fig 20 ELISA graph of GSK3.

Result

The data indicate a substantial presence of protein in BC, as evidenced by the prominent yellow coloration observed in the result images. The results and the calibration curve allows us to find out binding affinities and optimal dosage of our peptides with targeted receptors. In addition to the visual confirmation, the quality of the standard curve is optimal. Specifically, the R² value for PI3K was measured at 0.9937, for ANP32A at 0.9935, and for TrkB at 0.9975, which collectively support the robust use of BC in these experiments. Also, under 0.12062 mg/mL of BC protein concentration, the binding concentration of PI3K was at 1.899175069 ng/mL , ANP32A at 0.151433472 ng/mL, and TrkB at 0.058789625 ng/mL.

The findings for FT were consistent with those for BC. Strong yellow tones appeared in the FT result images, suggesting a notable protein presence as well. Furthermore, the standard curves achieved in FT tests were of excellent quality. For PI3K, the R² value reached 0.9718, for NOS it was 0.991, and for GSK3 the value stood at 0.9972, indicating FT had successful results in all three experiments. Under 0.12062 mg/mL of FT protein concentration, the binding concentration of PI3K was at 1.938588 ng/mL ,NOS at 0.592933444 µmol/mL, GSK3 at 12.78571429 pmol/L.

Summary of key findings

1. The BC fusion peptide showed consistently positive results across gel electrophoresis, SDS-PAGE, and ELISA assays, confirming its effective expression and functional activity.
2. ELISA tests on the FWT protein revealed no measurable color development and had a poor standard curve quality, indicating insufficient protein expression and a lack of functionality.
3. The redesigned FT protein demonstrated successful expression and functionality in all conducted assays, confirming the improved performance of the modified construct.

Based on these findings, we critically evaluated our initial approach and made strategic modifications to optimize the project’s outcomes. We shifted our focus toward improving protein design and expression. This iterative process allowed us to address initial shortcomings and refine our experimental strategy for better performance and reliability. For more comprehensive information about our project, please visit our engineering page, which provides a detailed overview of designing, building and testing of our eye drop.