Results

Biosensor Results

Probiotic Results

Lead Assay Results

The goal of the lead assay was to have a colorimetric assay that we could use to measure how much lead L. rhamnosus and B. subtilis was actually removing from water. It was needed because other lead-water testing uses equipment we do not have access to and sending samples for testing is both expensive and not recommended because we are intentionally putting bacteria in our samples. The assay was read in a plate reader in a 96-well plate. The absorbance was the result of interactions between glutathione, 20nm gold nanoparticles, and lead. The solution started a light pink color, and as an increasing amount of lead was present the solution would change to a darker purple-blue color. Before deciding that this assay would not be appropriate for our project, we did extensive assay development experiments to try to address its variability. We tried different protocols, optimized wavelength on available machinery for water, MRS, and LB, optimized GSH concentration for water, MRS, and LB, optimized pH of the solution and of the phosphate buffer for water, MRS, and LB, tried various lead concentrations, optimized type of gold nanoparticles, optimized how dilutions were made, optimized making of the GSH solution, and optimized reading time frames. In addition to this, we also found that readings were more accurate when the assay was done row by row, the gold nanoparticles were kept cold, and when the GSH and gold nanoparticles were added within 20 seconds of one another. We tried vortexing the samples before reading them; we double and triple checked the math for each dilution, and we considered doing a standard curve each time as if it were a Bradford Assay. Despite all of our work, the standard curve that was developed was not stable enough to accurately determine the concentration of known-unknown samples, and the assay could not be used in our project.

One of our standard curves can be seen below:

This graph shows one of our preliminary standard curves. Absorbance is on the y-axis, and lead concentration in parts per billion is on the x-axis. It shows that as the lead concentration increases so does the absorbance. Four samples were read per point on the graph, and the average of the four was used in the graph. The error bars on this graph looked very promising because of the limited overlap.

This is the same graph as the one above. This shows one of the reasons this assay was proved to be ineffective for our project. Instead of averaging all four of our samples per concentration, we averaged the three points that were closest for each lead concentration. The error bars increase because the difference in absorbance is not significant enough between the different lead concentrations. Similar results were seen throughout our assay development. This graph shows why we were unable to place other samples of known lead concentrations on the standard curve without looking at the concentrations.

Lead Test Kit Results

After our failed attempt at creating a DIY lead assay, we chose to use the Hach LeadTrak kit to quantify the amount of lead that L. rhamnosus and B. subtilis could remove from water. The protocol involves running a sample through a Fast Column Extractor, adding a lead indicator to the eluent, and measuring the OD477 of the sample. After creating a standard curve using the lead standard solution provided in the kit that plots lead concentration (up to 150 ppb) against absorbance at OD447, the lead concentration of any sample can be determined. Our standard curve is shown below:

Using our standard curve, we were able to quantify the amount of lead absorbed by our “force-evolved” B. subtilis culture over a 24-hour period. As previously mentioned, B. subtilis was streaked on a LB-agar plate that contained 2,000 ppm of lead. We hypothesized that because the B. subtilis colonies on this plate had grown in the presence of an extremely high quantity of lead, they would would have evolved to bind more lead than colonies that had grown without any lead (control colonies). In order to determine whether the “force-evolved” culture could in fact bind more lead that the control culture, we measured the lead absorbance over time for cultures made from each of the two colonies. The colonies used to produce the control culture were taken from a standard LB-agar plate streaked with B. subtilis. The second, “force-evolved” culture was produced using colonies from the 2,000 ppm lead LB-agar plate. Each culture was grown and sampled in a accordance to the Lead Test Kit Protocols provided on the Experiments page.

The amount of lead bound to B. subtilis for each culture over time is shown below:

In order to ensure that the difference in lead binding over time was not a result of differences in the number of cells present in each culture (determined by the OD600 of the culture), we control for this by dividing the lead concentration by the OD600 in the graph below:

We believe that the large amount of error associated with the first time point is due to the fact that the OD600 of the cultures at this point is still very low. Once the cells reach log phase (hours 4 and 24), we can see an increase in lead binding per OD overtime with minimal error, suggesting that the “force-evolved” culture could have bound more lead over time than the control culture.