Week 10 HW: Advanced Imaging & Measurment Technology

Homework: Final Project

For my project, I want to measure the activity of the CotA laccase from Bacillus subtilis (whose gene is inserted into my pETite‑based plasmid) against PAH compounds using ABTS as a colour‑indicating substrate. I will measure the initial reaction rate of CotA, expressed as the change in absorbance over time, under different conditions, such as pH and temperature, to determine the enzyme’s optimal working range.

I would also like to measure the half‑life and stability of CotA in a paint‑like formula by embedding cultured CotA‑expressing cells in a water‑based gel or coating to then sample at regular intervals and quantify any residual activity through ABTS assays. Finally, I will record colour‑change metrics of the ABTS PAH‑surrogate solution over time, using percentage colour loss as a quantitative readout of degradative activity. This will help characterise the functional performance of the system in conditions relevant to a PAH‑degrading paint.

Considering PAH are not LBS1 safe, I will be using ABTS, a synthetic water‑soluble compound that laccases (like CotA) oxidise to a coloured radical as a PAH substitute. After mixing my pETite‑CotA plasmid with BL21(DE3) competent cells, performing heat shock and plating on LB kanamycin to grow a colony. I can then proceed to scaling up the culture to have enough E.coli to proceed with the following tests:

Reaction Rate of CotA Laccase - Change in absorbance over time:

2) Prepare the solution: dissolve ABTS in 100 mM potassium phosphate buffer (achieved by mixing Potassium phosphate monobasic and dibasic in water).

3) Centrifuge the culture to pellet the cells and pipette away the surplus (LB and kanamycin) to obtain only the bacterial cells.

4) From the obtained cells, I will then resuspend the pellet in a smaller volume of buffer to create a cell suspension.

5) I will then measure the ABTS assay of the cell suspensions of my prepared bacterial samples at different temperatures and pHs by using a 96-well plate reader or UV-Vis spectrophotometer, depending on lab availability.

6) I will measure the change in absorbance at 420nm over time during the ABTS oxidation assay.

7) I will calculate the initial rate as ∆A₄₂₀·min⁻¹, which serves as a quantitative measure of CotA activity under each condition.

8) Additionally, adding copper to the reaction will enhance the activity of CotA Laccase.

Half‑life and stability of CotA‑expressing cells embedded in a paint‑like gel

References

For all three protocols, the core instrument is a UV‑Vis spectrophotometer (or a microplate reader with absorbance capabilities) to measure A₄₂₀ during ABTS‑based laccase assays. For the settings, I will set the wavelength at 420 nm (ABTS has the highest absorbance point). I will then create a ‘blank’ cuvette to calibrate it (buffer + ABTS with no CotA cells) and place it first in the spectrometer. Place each sample in a cuvette and place those in the spectrophotometer one by one. Calculation details are explained in the bullet point above.

Additionally, to relate to the lecture, my aim 3 for my project aims to develop a field‑ready bioremediation paint by evaluating CotA performance in a real‑world‑like coating matrix and validating its safety profile. Toxicity validation would focus on verifying that degradation yields safe byproducts rather than more hazardous compounds, a concern highlighted by recent studies showing that PAH transformation products could be toxic (Huizenga et al., 2025). In this context, mass spectrometry (e.g., LC‑MS/MS) would be used to identify and monitor the formation of degradation products (Nakken et al., 2025).

References