In order to develop a kit to test soil nutrients that could work for farmers and gardeners, it is necessary to go through the steps of design, build, test, learn, rebuild. We have been working these steps as we try to design something that is simple, cost-effective, and accurate.
We were able to show that the copper-inducible bla gene worked. By adding increasing concentrations of copper sulfate solution, we were able to observe an increase in red color indicating that there was an increase in the expression of the bla gene. The bla gene codes for the Beta-Lactamase enzyme. When more copper is present, more Beta-Lactamase is produced which causes increased cleavage of its substrate, nitrocefin. When nitrocefin is cleaved, a red color is produced. We will show a graph demonstrating the increase in red color.
In the design steps, we had to make many choices regarding the basic characteristics of our constructs and kits. First, we had to decide which nutrients we wanted to attempt to measure. From reading and the interviews with farmers, we learned that there are three very important nutrients that are measured in soil: phosphate, nitrate, and potassium. In order to measure these nutrients with a biosensor like we planned to use, we needed to gene promoters that were sensitive to these nutrients. In our searching, we found the phosphate sensitive promoter for the gene, phoA, in E. coli. We found a couple possibilities for nitrate and nitrite promoters but there was not a simple potassium-sensitive bacterial promoter that we could find. Therefore, we focused on the phosphate, nitrate, and nitrite promoters. While investigating, we also found a gene that is controlled by a copper-inducible promoter. While this is not a nutrient that is often measured when looking at soil quality for farming, it is certainly an important micronutrient. Thus, we included a copper-inducible promoter in our experiments.
While building the constructs, we also had to decide that reporter we wanted to use. We settled on a colorimetric reporter. A fluorescent reporter could be relatively simple but it would be difficult for an average person outside of a science lab to observe or measure fluorescence. Other reporters like lacZ were too large to synthesize in our construct so we settled on the bla gene which codes for Beta-lactamase.
When building the phosphate construct, we also realized that we need to make it more complex because phosphate negatively regulates this promoter. More phosphate reduces the expression from the phoA gene. Therefore, we had to combine this negative regulation with the negative regulator of the lac operon. By putting the phoA promoter on the gene for the Lac repressor, we were negatively regulating a negative regulator. Thus, we could create positive regulation when phosphate was present. So, with all of the constructs we would expect to see an increase in color when the nutrient concentration increased.
There were multiple tests that had to be performed to see if the constructs we designed worked but there were also tests to figure out how we would test for nutrients from soil. First, we put the constructs in strains of E. coli and tested their ability to sense the different nutrients by using solutions of each nutrient. We found that the phosphate and copper-inducible promoter constructs worked the best. We did not see encouraging results from the nitrate and nitrite-inducible promoters initially. While there are more experiments that we could attempt with the nitrate and nitrite promoters, in order to focus our efforts and save time, we moved forward with the phosphate and copper promoter strains.We tested many variables with the phosphate and copper-inducible constructs including the concentration of bacteria, concentration of nutrient solution, concentration of the substrate for the Beta-lactamase reporter, and the amount of time we waited before measuring the color. This allowed us to develop a rough protocol for this process.
We also tried to determine how much dirt to use for the test, how we would create a solution in which we would introduce the E. coli containing our constructs, and how we would be able to observe the color change. For this we decided on a rough amount of soil to use, a specific buffer to use to wet the soil, how to filter the solution after determining how long to allow the soil to sit in the buffer, and then how to incorporate this solution into the protocol we developed for the constructs.
Overall, we have developed a rough method for measuring phosphate and copper concentration.
As described in the testing phase, we learned from each of the initial tests we performed. While testing nutrient concentrations, we had to determine what levels of nutrient the constructs were able to measure and if this was relevant to potential amounts of nutrients found in soil. We had to think about whether the methods we were using to perform these protocols were something that an average person could use. Most importantly, we wanted to see if the measurements were accurate and reproducible. We have been able to find the answers to many of these questions but this still a work in progress.
As we perform the tests and get results, this informs us for the next set of experiments. We were able to learn that we were using too much of the enzyme substrate initially. Therefore, we reduced that amount. The highest concentrations of copper likely killed the E. coli we were using but we learned that lower concentrations worked fine. We also added steps to the protocol for the kit to make it easier to use and produce better results.