Overview

The WLC-Milwaukee iGEM team attempted to create test kits that could easily, affordably, and accurately measure the amount of specific nutrients in soil. The specific nutrients we attempted to measure were phosphate, nitrate, nitrite, and copper. The kit was based on strains of E. coli that contained plasmids with nutrient-inducible promoters controlling the bla gene. This gene codes for Beta-lactamase which is an enzyme capable of cleaving a substrate that produces a red color. Therefore, if the kit works correctly, an individual would be able to obtain a soil sample from any location and use the kit to measure the amount of phosphate, nitrate, nitrate, or copper based on the amount of red color produced when the tests are performed.

Do our constructs work?

The first experiment we wanted to perform was to test whether we saw red color when the E. coli strains containing our various nutrient-inducible genes for Beta-lactamase were provided the specific nutrients and the Beta-lactamase substrate, nitrocefin. To test this we used dilutions of sodium phosphate buffer, Sodium nitrate, sodium nitrite, and copper sulfate.

Figure 1 – Phosphate construct with dilutions of sodium phosphate buffer

This figure shows tubes containing the E. coli cells containing a phosphate-inducible bla gene that were provided dilutions of sodium phosphate buffer starting with 20 mM sodium phosphate (final concentration) and progressing to the right with 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.312 mM, and 0.156 mM sodium phosphate. The third tube from the right was E. coli with 20 mM sodium phosphate but no nitrocefin added. The second tube from the right was an E. coli strain with a different plasmid that constitutively expressed bla. The tube on the far right was a strain of E. coli that received nitrocefin but did not have a plasmid with the bla gene so no Beta-lactamase was produced.

It was apparent from this first experiment that Beta-lactamase was being expressed from our construct but there was little difference in the red color between the tubes with the varying concentrations of phosphate. This could be due to too much nitrocefin being added or too much E. coli.

Figure 2 – Nitrate-inducible construct with dilutions of sodium nitrate

This figure shows tubes containing the E. coli cells containing a nitrate-inducible bla gene that were provided dilutions of sodium nitrate solution starting with 200 mM sodium nitrate (final concentration) and progressing to the right with 100 mM, 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.12 mM, and 1.56 mM sodium nitrate. The third tube from the right was E. coli with 200 mM sodium nitrate but no nitrocefin added. The second tube from the right was an E. coli strain with a different plasmid that constitutively expressed bla. The tube on the far right was a strain of E. coli that received nitrocefin but did not have a plasmid with the bla gene so no Beta-lactamase was produced.

While there was a slight color change in the tubes, there was not nearly the change that we saw with the phosphate-inducible construct. Many variables could have been tested with these strains, especially oxygen concentration, since the promoter for the nitrate-inducible gene in E. coli from which this was taken works better in anaerobic conditions. We only tested aerobic conditions. Due to limitations in time, we did not continue to test the nitrate-inducible strain.

Figure 3 – Nitrite-inducible construct with dilutions of sodium nitrite

This figure shows tubes containing the E. coli cells containing a nitrite-inducible bla gene that were provided dilutions of sodium nitrite solution starting with 20 mM sodium nitrite (final concentration) and progressing to the right with 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.312 mM, and 0.156 mM sodium nitrite. The third tube from the right was E. coli with 20 mM sodium nitrite but no nitrocefin added. The second tube from the right was an E. coli strain with a different plasmid that constitutively expressed bla. The tube on the far right was a strain of E. coli that received nitrocefin but did not have a plasmid with the bla gene so no Beta-lactamase was produced.

Similar to the nitrate experiments, there was not a large change in color for the nitrite-inducible construct. While there did appear to be more color change for the nitrite than nitrate, it still was not enough to warrant spending more time. Much like the nitrate-inducible strain, we could change variables such as oxygen concentration, nitrocefin concentration, and E. coli concentration to see if we could increase color production.

Figure 4 – Copper-inducible construct with dilutions of copper sulfate

This figure shows tubes containing the E. coli cells containing a copper-inducible bla gene that were provided dilutions of copper sulfate solution starting with 20 mM copper sulfate (final concentration) and progressing to the right with 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.312 mM, and 0.156 mM copper sulfate. The tube on the far right was E. coli with 20 mM copper sulfate but no nitrocefin added.

These results were very promising. While the three tubes with the highest concentration of copper sulfate were blue or green in color suggesting the E. coli were either greatly inhibited or killed, these were concentrations well above expected concentrations of copper in soil. The lower five concentrations of copper sulfate showed significant red color and there appeared to be a slight change in the red color from the highest copper sulfate concentration to the least copper sulfate.

While additional tests were performed with the phosphate and copper constructs, it was determined that we would move forward with a trial run of the entire soil testing procedure with the copper construct to see if we could detect copper in soil. To do this, we needed to start with soil sample preparation and then test the solution acquired from this soil preparation with the E. coli containing the copper-inducible construct.

How does the test kit work?

Figure 5 – Soil sample

A soil sample was acquired from behind Generac Hall on the Wisconsin Lutheran College campus. Soil was added and loosely packed in a 50 ml conical tube up to the 40 ml line.

Figure 6 – Soil sample with Tris buffer

Approximately 25 ml of 10 mM Tris buffer pH 7.5 was added to the soil in the conical tube and vortexed vigorously for 30 seconds. This allowed the soil to become saturated with buffer with the expectation that at least some copper in the soil would dissolve in solution.

Figure 7 – Filtration of the soil sample

The buffer liquid from the soil sample was then passed through filters to reclaim a clear liquid. First, an initial filtration step took place (on the left) to remove the largest soil particles. Then a second filtration step took place (on the right) with a 0.45-micron syringe filter to remove small soil particles.

Figure 8 – Clear solutions were acquired after filtration

After the filtration steps were completed, clear solutions were reclaimed from the soil samples. These solutions should contain copper from the soil that we can then measure with the copper-inducible E. coli strain.

Figure 9 – Results of a test with the copper-inducible E. coli strain

The results from our initial test were positive. We set up four standards with known concentrations of copper that can be compared to the soil sample solution. There were also two controls including one in which no nitrocefin is added to ensure there was no red color coming from the solutions. The other control received Tris buffer instead of copper sulfate or soil sample.

As we can see in these results, there are varying amounts of red color depending on the amount of copper provided in the standards. The encouraging result is that the amount of red color produced from the soil sample falls within the range seen in the standards. One problem is that there is a significant amount of red color being produced when there is no copper sulfate or soil sample provided. This suggests Beta-lactamase is being produced even when there is not much copper in the solution. This could be due to residual copper in the E. coli growth media or leaky expression from the copper-inducible promoter.

While there is a difference in the amount of red color present in the standards, it would be much clearer if there was a greater difference. Some of the problems may be with the background red color mentioned above. Further experimenting with different concentrations of E. coli and nitrocefin could alleviate some of these issues.

Future Research

The results for the copper-inducible E. coli strain shown above are very promising. It demonstrates that Beta-lactamase is being produced from the construct while more experiments need to be performed to fine-tune the amount of Beta-lactamase being produced.

There are also additional experiments that need to be performed with the soil sample. We need to perform chemical analysis to determine the actual concentration of copper in the soil sample so we can determine if our test is accurate in its quantification of copper. There are also many other buffers we could use to see if others are better at collecting copper from the soil. Variables such as incubation times and temperatures could also be tested.

Furthermore, the other nutrient-inducible constructs could be tested more to determine if they are capable of measuring nutrient concentrations in soil. Each construct will require its own set of experiments to determine its optimal conditions and accuracy. Given more time, we would have liked to test more of these conditions.