iGEM 2022 — Team wlc-milwaukee

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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Engineering

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.

![Design](https://static.igem.org/mediawiki/2021/d/d3/T--WLC-Milwaukee--designeng.png)

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.

![Build](https://static.igem.org/mediawiki/2021/2/24/T--WLC-Milwaukee--build.png)

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.

![Test](https://static.igem.org/mediawiki/2021/1/1d/T--WLC-Milwaukee--test.png)

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.

Experiment

# Example Experiment

This project required many basic synthetic biology techniques. See below for the techniques we used, reagents required, and a brief protocol. Note that all techniques require basic microbiology tools (pipettes, tubes, plates, incubator, etc.)

1. Five constructs of plasmid were designed to be utilized as molecular biosensors for nutrient levels within soil. All constructs were synthesized in the medium copy number plasmid “pTwist-kan” These constructs include:

a. Copper-inducible construct
b. Nitrate construct
c. Nitrite construct
d. Phosphate construct with fully functioning Lac operon
e. Phosphate construct with LacI degradation tag

2. Transformation

Added to microcentrifuge tubes:
a. 50 uL DH5a electrocompetent *E. coli* cells
b. 5 uL Synthesized plasmid

i. Add solution to electroporation cuvette

c. Pulse
d. Quickly add 1 mL of LB broth to the cuvette
e. Transfer solution from cuvette to microcentrifuge tube
f. Incubate at 37 C for 30 min
g. Centrifuge at 13.7 x g for 1 min
h. Decant LB
i. Add 50 uL of fresh LB broth, resuspend pellet
j. Spread 100 uL on LB/ Kan agar
k. Incubate at 37 C overnight

3. Qualitative Color Change:

a. Multiple dilutions of Copper, nitrate, and phosphate buffers
b. Grew 5 ml overnight cultures of *E. coli* strains contain the 5 constructs
c. Five 1:10 dilutions of 0.1 M CuSO4, 1M NaNO3, and 0.1M NaNO2

i. NaNO2: 0.1M, 0.05M, 0.025M, 0.0125M, 0.00625M
ii. NaNO3: 1M, 0.5M, 0.25M, 0.125M, 0.0625M
iii. CuSO4: 0.1M, 0.05M, 0.025M, 0.0125M, 0.00625M
iv. NaH2PO4: 0.1M, 0.05M, 0.025M, 0.0125M, 0.00625M

d. 500 uL of each culture added to the corresponding 125 uL buffer
e. Incubated 5 minutes
f. 4 microliters of 10 mg/ml Nitrocefin were added to all tubes
g. Pictures were taken at 5 and 15 minutes after the addition of nitrocefin

i. Control: *E. coli* without plasmid
ii. Control: No nitrocefin added

h. Different ratios of the nitrocefin were then experimented with

4. Light Spectrophotometry:

a. Spectrophotometer ran at wavelength of 600nm

i. To measure concentration of bacteria

b. Absorbance readings were taken for different dilutions of copper sulfate and sodium phosphate

i. 4 dilutions – 1x10^-3,1X10^-4 M, 1X10^-5 M, 1X10^-6 M
ii. Control: No nitrocefin
iii. Control: No buffer

c. Later this was repeated with extracting nutrients out of dirt as well

i. 4 dilutions – 1x10^-3,1X10^-4 M, 1X10^-5 M, 1X10^-6 M
ii. Control: No nitrocefin
iii. Control: No buffer
iv. 10 minute dirt water samples
v. 25 minute dirt water samples

5. Soil Sample Preparation:

a. A 50 ml conical tube was used to acquire 40 ml of loosely packed soil
b. 25 ml of 10 mM Tris pH 7.5 was added to the soil sample

i. The sample was vortexed for 30 seconds

c. A funnel with filter paper was placed in a new 50 ml conical tube
d. The soil and buffer solution was poured into the funnel with filter paper to remove large soil particles

i. Liquid was allowed to collect until 5 ml was present

e. A 0.45 micron syringe filter was attached to a 10 ml syringe and placed in a 15 ml conical tube
f. The 5 ml soil solution was poured in the syringe and passed through the syringe filter to remove any remaining large soil particles
g. The solution that was obtained from the filtrations steps was used for testing with the *E. coli* strains containing the nutrient-inducible constructs

6. UV-Vis Spectrophotometry (for copper experiment)

*E. coli* was prepared similar to what is described in the Quantitative color change experiments above with some modifications

a. 50 ml cultures of *E. coli* was prepared the day before the experiments were to take place
b. The following day 4 ml of *E. coli* containing the copper-inducible construct were added to seven tubes
c. All seven tubes were centrifuged at 4500 rpm for 5 minutes
d. The supernatant was removed from the tubes
e. The *E. coli* cells were resuspended in 2 ml of 10 mM Tris pH 7.5 buffer
f. 0.5 ml of the 1x10-3,1X10-4 M, 1X10-5 M, 1X10-6 M copper sulfate solution were added to four tubes
g. To one tube, 0.5 ml of 1x10-3 M copper sulfate was added but no nitrocefin was added (“no nitrocefin” tube)
h. To one tube, 0.5 ml of 10 mM Tris buffer was added
i. To one tube, 0.5 ml of the soil sample solution was added
j. To all of the tubes except for the “no nitrocefin” tube, 4 microliters of 10 mg/ml nitrocefin was added
k. All tubes were allowed to incubate at room temperature for one hour
l. After incubation, 1.2 ml of each mixture was pipetted into a microcentrifuge tube
m. All of the tubes were centrifuged for 1 minute at 13,300 g in a microcentrifuge
n. One ml of liquid from each tube was transferred to a cuvette
o. Cuvettes containing these solutions were analyzed by the UV-Vis spectrophotometer at wavelengths from approximately 200 nm to 820 nm
p. Readings from the spectrophotometer were transferred to an excel file and the data was analyzed

Results & Demonstration

# 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

![Phosphate dilutions](https://static.igem.wiki/teams/4366/wiki/results/results1.jpg)

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

![Nitrate dilutions](https://static.igem.wiki/teams/4366/wiki/results/picture2.jpg)

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

![Nitrite dilutions](https://static.igem.wiki/teams/4366/wiki/results/picture3.jpg)

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

![Copper dilutions](https://static.igem.wiki/teams/4366/wiki/results/picture4.jpg)

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

![Soil Sample](https://static.igem.wiki/teams/4366/wiki/results/picture5.jpg)

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

![Soil w/ Buffer](https://static.igem.wiki/teams/4366/wiki/results/picture6.jpg)

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

![Filtration](https://static.igem.wiki/teams/4366/wiki/results/picture7.jpg)

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

![Filtration](https://static.igem.wiki/teams/4366/wiki/results/picture8.jpg)

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

![Copper test](https://static.igem.wiki/teams/4366/wiki/results/picture9.jpg)

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.