Difference between revisions of "Team:WPI Worcester/Results"
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| + | <h3> Lead Assay Results</h3> | ||
| + | <p> | ||
| + | The goal of the lead assay was to have a colorimetric assay that we could use to measure how much lead lactobacillus was actually removing from water. It was needed because other lead-water testing uses equipment we do not have access to and sending it 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. Some of our graphs can be seen below: | ||
| + | </p> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/17/WPI_Worcester_2017_Lead_Assay_Graph_with_Outliers.png" alt="Lead Assay Graph with Outliers" style="width:800px;height:428px;"> | ||
| + | <p> 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. | ||
| + | </p> | ||
| + | <br><br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/0/06/WPI_Worcester_2017_Lead_Assay_Graph_without_Outliers.png" alt="Lead Assay Graph without Outliers" style="width:800px;height:500px;"> | ||
</div> | </div> | ||
Revision as of 15:12, 13 October 2017
Project
Parts
Notebook
Safety
Human Practices
Attributions
Awards
Human Practices
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 lactobacillus was actually removing from water. It was needed because other lead-water testing uses equipment we do not have access to and sending it 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. Some of our graphs 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.
