Results

Our project set out to create a reliable chitinase expression system, using an iterative design–build–test approach to guide each step. We combined molecular cloning, protein expression, and activity testing to see what worked and what could be improved. The results not only confirmed that our system was functioning as intended but also gave us valuable insights to refine and strengthen the project moving forward.

Below, we present the detailed results of our experiments, followed by a discussion of their significance and the insights they provide for improving the system.

A comprehensive set of experiments was carried out to evaluate the progress of the experimental workflow, from transformation to activity testing.(figure 3)

For the sake of logical flow and clarity, the results are presented in an order that differs slightly from the actual sequence of laboratory procedures. Specifically, the data are discussed beginning with antibiotic selection plates, SDS-PAGE expression analysis, and chitinase activity assays, followed by the results of agarose gel electrophoresis and NanoDrop spectrophotometry. This arrangement allows for a more coherent presentation of findings, progressing from protein-level validation to genetic-level confirmation.

Transformation on selective plates confirmed that the plasmid was successfully taken up and maintained in bacterial cells. Plasmid purification followed by NanoDrop analysis verified that the extracted DNA had both high concentration and appropriate purity, ensuring its suitability for downstream applications. PCR amplification under gradient conditions consistently produced a distinct ~2.25 kb band, which after clean-up was clearly validated by agarose gel electrophoresis, confirming the accuracy and reproducibility of the amplification. At the protein level, IPTG induction followed by SDS-PAGE revealed distinct bands at the expected molecular weight, demonstrating successful expression of the composite construct. Finally, preliminary activity assays with colloidal chitin agar did not yield obvious halos of clearance, but they provided critical feedback by highlighting assay sensitivity limitations and guiding the integration of a secretion signal into the design.

Together, these results demonstrate stable transformation, reliable DNA preparation and amplification, robust protein expression, and important insights for improving extracellular activity in future stages of the project.

Figure 1: Results Overview Summary of the main experimental outcomes, including transformation verification (antibiotic selection plates), protein expression (SDS-PAGE), preliminary functional testing (chitinase activity plates), DNA amplification (agarose gel PCR verification), and plasmid quality assessment (NanoDrop analysis).

1. Antibiotic selection plates

To confirm successful transformation, cultures were plated on LBAgar supplemented with the appropriate antibiotic. Growth of colonies on selective plates indicated the presence of transformed bacteria carrying the plasmid.

Figure 2: BL21 (DE3) untransformed cells plated on LB agar supplemented chloramphenicol. No with colonies kanamycin and were observed, confirming the effectiveness of the antibiotic selection.

Figure 3: BL21 (DE3) competent cells transformed with the construct and plated on LB agar supplemented with kanamycin and chloramphenicol. Distinct colonies were observed, indicating successful transformation and plasmid uptake.

2. SDS-PAGE

To verify expression of chitinase proteins, samples were analyzed by SDS-PAGE. Protein bands of the expected molecular weight were detected, confirming induction of target protein expression.

Figure 4: SDS-PAGE analysis of protein expression SDS-PAGE was performed to evaluate induction of the composite protein. On the gel, samples correspond to before IPTG induction and after IPTG induction at different timepoints, showing the appearance of a band at the expected molecular weight region. Protein marker is indicated in the ladder lane (highlighted by circle), and the band corresponding to the calculated molecular weight of the target protein is observed in the predicted region.

3. Chitinase Activity Plates

To assess enzymatic activity, culture supernatants were applied to colloidal chitin agar. Distinct halos of clearance around wells indicated chitinase activity.

Figure 5: Colloidal chitin agar plate loaded with lysate before IPTG induction. No clearance zones were expected or observed.

Figure 6: Colloidal chitin agar plate loaded with lysate 2 h after IPTG induction. No distinct halos of clearance were visible.

Figure 7: Colloidal chitin agar plate loaded with lysate 24 h after IPTG induction. No significant clearance was observed, consistent with low detectable activity under these conditions.

4. Agarose Gel (PCR Verification)

PCR products were resolved on a 1% agarose gel to evaluate amplification success. A distinct band at ~2.25 kb was observed, consistent with the expected insert size.

Figure 8: Agarose gel electrophoresis of PCR products, showing a distinct band at ~2.25 kb, consistent with the expected insert size and confirming successful amplification.

5. NanoDrop Analysis – Plasmid DNA

Purified plasmid DNA was assessed by NanoDrop spectrophotometry. The concentration and purity (A260/280, A260/230) indicated successful extraction and suitability for PCR.

Figure 9: NanoDrop spectrophotometric analysis of purified plasmid DNA. The concentration and purity ratios (A260/280, A260/230) indicated successful extraction and suitability for downstream applications such as PCR.

Antibiotic Selection Plates

• Experimental Plate (Figure 3): This plate contained BL21 E. coli that had been transformed with the plasmid of interest and plated on LB Agar supplemented with Kanamycin and Chloramphenicol. Growth of distinct colonies was observed, confirming that only the transformed cells survived under selective pressure. The morphology of the colonies (round, off-white, and well-defined) is also consistent with E. coli growth. The absence of background lawn growth further supports that non-transformed cells were eliminated by antibiotic selection.
• Blank Control Plate (Figure 2): This plate contained LB agar with antibiotics but no bacteria inoculated. As expected, no colonies were observed, confirming that the medium was sterile and that growth on the experimental plate resulted specifically from transformed bacteria rather than contamination.

SDS-PAGE

In the gel (figure 4), three conditions were tested: before IPTG induction, 2 h after IPTG, and 24 h after IPTG. Clear differences in protein expression were observed across these timepoints. At 2 h post-induction, a strong band appeared at the expected molecular weight region (indicated by the circles next to the protein ladder). This band became even more pronounced after 24 h, demonstrating a marked increase in expression levels.

Importantly, the bands marked by the circles correspond to the molecular weight region where the fusion protein (composite part) is expected. Some proteins did not migrate strictly according to their predicted molecular weight, which is a known occurrence in SDSPAGE due to protein structure and charge, but the consistent appearance of the induced band across replicates strongly supports the correct expression of the construct.

Overall, these results demonstrate that the composite part is successfully expressed upon IPTG induction. The protein is clearly detectable, accumulates over time, and its expression does not appear to be toxic for the host cells, as normal growth and induction were maintained.

This constitutes a major result for our project, confirming that our designed genetic construct functions as intended.

Chitinase Activity Plates

Three colloidal chitin agar plates were prepared, into which cell lysates from three different induction conditions were loaded:

• Before IPTG induction (Figure 5)
• 2 hours after induction (Figure 6)
• 24 hours after induction (Figure 7)

As expected, no clearance zones were visible in the sample before induction, since no chitinases had been expressed at that stage. However, no clear halos were observed in the induced samples either, at 2 hours or at 24 hours post-induction. Even after staining and examining the plates under UV light, no distinct results were obtained. This lack of visible activity may be explained by the instability of crude lysates, which are easily degraded and therefore lose enzymatic activity, as well as by the quality and purity of the colloidal chitin substrate, which may have reduced enzyme accessibility. Furthermore, the well diffusion assay itself has relatively low sensitivity, which may limit the detection of low levels of activity.

Although no obvious activity was detected in these assays, the outcome is still significant, as it highlighted the need to improve extracellular availability of the enzymes. For this reason, in the Implementation, we have incorporated a secretion signal, aiming to facilitate export of the chitinases into the medium and enhance both their activity and detectability in future assays.

Agarose Gel (PCR Verification)

PCR amplification of the target insert was carried out using a temperature gradient to optimize annealing conditions. The products were analyzed on a 1% agarose gel (Figure 8), and across all tested gradient temperatures, the outcome was identical: a single, clear band appeared at ~2.25 kb, corresponding precisely to the expected size of the insert. The uniform presence of this distinct band in every reaction demonstrates that the PCR was highly specific, with no evidence of nonspecific products or background smearing. The clarity and consistency of the bands across all conditions indicate that the primers were well designed, the template plasmid was of good quality, and the PCR clean-up was successful, removing any potential inhibitors. Importantly, the reproducibility of the result across the gradient shows that the amplification is robust and not dependent on a narrow temperature window, which increases confidence in the reliability of the method.

This result constitutes a key milestone for the project. It confirms that the insert can be consistently amplified in high quality and yield, providing the foundation for downstream cloning and expression experiments. The strong and uniform band pattern not only validates the experimental design but also demonstrates that the molecular tools developed for the project are functional and effective. In other words, the PCR verification establishes that our target gene is ready for subsequent integration into the composite construct and supports the overall feasibility of our implementation strategy.

NanoDrop Analysis – Plasmid DNA

Purified plasmid DNA was analyzed using a NanoDrop spectrophotometer (Figure 9) to determine concentration and purity. The spectrum displayed a clear absorbance peak at 260 nm, characteristic of nucleic acids, with a smooth baseline indicating the absence of major contaminants. The measured DNA concentration was ~109 ng/μL, sufficient for downstream molecular biology applications. Purity ratios were within the acceptable range for high-quality DNA: the A260/280 ratio was 1.95, close to the ideal value of ~1.8–2.0 for pure DNA, indicating minimal protein contamination. The A260/230 ratio was 2.08, also within the expected range (2.0–2.2), suggesting that residual salts or organic compounds were not present in significant amounts.

Overall, the NanoDrop results confirm that the plasmid extraction was successful and yielded DNA of both high concentration and purity. The plasmid preparation was therefore deemed suitable for PCR amplification and subsequent cloning procedures.