General Contributions
We are using Beta-lactamase to create a field kit for farmers to measure the copper and phosphate contents of their soil. Because we now have these plasmids, the opportunity to further explore other nutrient contents in the soil is now a possibility. It is our hope that future iGEM teams will be able to further explore the implications of soil health through the use of our parts.
Furthermore, we aim to have contributed to the field of agriculture by making soil testing more accessible. Through our interviews with a variety of farmers, we learned what makes soil testing kits particularly useful. We learned that being able to ensure the health of soil from year to year is a crucial part of growing the food we eat. Throughout all of our interviews, we gained an insight into the amount of effort that goes into maintaining healthy soil. We hope that our research and the synthesis of our soil testing kit will help future iGEM teams further discover the intricacies of how to measure the health of soil, and use it to not only help farmers, but also communicate the importance of soil health to all.
Troubleshooting
As is natural to the process of laboratory work, roadblocks arise that require researchers to investigate the cause of the issue and modify their original laboratory plans accordingly. The WLC-Milwaukee lab group encountered some obstacles concerning nitrocefin and color changes that required adjustments to our original experimental plans.
Within the initial experiments evaluating color change at various phosphate and copper concentrations, the control, containing no buffer, unexpectedly appeared to take on the same color as the non-control solutions. This suggested that the buffer made no difference in the color of solutions. This phenomenon led us to believe that the concentration of nitrocefin was too high. If this was true, beta-lactamase was causing the red color and disallowing us from observing a difference among different concentrations of buffer. After arriving at this explanation, we conducted more experiments to determine more appropriate nitrocefin concentrations.
In the phosphate experiments to determine appropriate nitrocefin concentrations, we had difficulty distinguishing between buffer and non-buffer tubes. Since we were using E. coli strains in the media in which they were grown, we believed that there was phosphate in the media itself that was altering our results.This finding led us to conduct additional experiments in order to quantify the color changes that occurred.
Following an experiment evaluating copper samples, phosphate samples, and Tris buffer mixed with soil, another obstacle was encountered when no red color was observed in the resulting solutions. Given that the process had been similar to previous experiments yet had produced different results, we were led to hypothesize that the nitrocefin had become ineffective due to thawing and refreezing it multiple times. Because of this, a new tube of nitrocefin was used in subsequent experiments. During experiments in which the new nitrocefin was used, there in fact was a color change.
The modifications made during the course of lab work provide several takeaways that will be useful to those performing similar experiments. The concentration of nitrocefin, phosphates within media, and the process of freezing and thawing nitrocefin have the potential to affect results. However, the adjustments made over the course of the lab work for this project have been demonstrated to improve results. Therefore, these adjustments can be utilized in other projects when investigators run into similar issues.
Parts
WLC-Milwaukee contributed five constructs of plasmid that can be used as biosensors to evaluate the presence of nutrients within soil. These include a copper-inducible construct, a nitrite construct, a nitrate construct, a phosphate construct with a fully-functioning lac operon, and a phosphate construct with a lacl degradation tag.