iGEM 2023 — Team wlc-milwaukee


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{% block title %}Engineering Success{% endblock %}
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Engineering success can be defined by following the engineering design cycle:

Design → Build → Test → Learn

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In order to develop a test that could be used to determine the concentration of bioavailable
phosphate in soil, we needed to go through the steps of design, build, test, learn, retry. We have been
working through these steps to design a testing procedure that is simple, cost-effective, and accurate.

For our project last year, we designed a construct that used the Lac repressor to control the
expression of the *bla* gene in response to phosphate levels. The *bla* gene codes for Beta-lactamase which is
an enzyme that can produce a red color when a substrate called nitrocefin is added. One significant problem
that we found was that there was a lot of background red color suggesting that there is Beta-lactamase being
produced independently of the phosphate-controlled regulation.

It is known from studying the lac operon that the Lac repressor allows leaky expression of the
genes it is regulating so we wanted to try using a different repressor that may provide more tight
regulation of the *bla* gene. This led to us trying TrpR which is a negative regulator of the *trp* operon. This
repressor binds more tightly to the *trpL* operator sequence so we thought this might help remove some of the
background red color that makes our data difficult to interpret.

---

### Design

In the design stage of our project we needed to decide which regulators would potentially work
the best to control the expression of the *bla* gene in response to phosphate concentrations. We knew that we
were going to use the *phoA* promoter because it is sensitive to phosphate levels and there are very few
alternatives currently known. The *phoA* promoter is tricky, however, because it works the opposite of how we
would like it to work. The *phoA* promoter turns expression off when there are high levels of phosphate and
turns expression on when there are low levels of phosphate. To account for this mechanism, we put the *phoA*
promoter in control of a negative regulator. This way, if there are high levels of phosphate, it would
reduce the amount of the negative regulator which would turn on the expression of whatever the negative
regulator was regulating.

As mentioned above, we initially used the Lac repressor as our negative regulator but this proved
to be too leaky. We considered using AraC from the arabinose operon since this is a tightly regulated operon
but AraC can work as both a positive and negative regulator so it would lead to a more complicated system.
We settled on TrpR from the *trp* operon so we placed the *trpR* gene under the control of the *phoA* promoter and
placed the *trpL* operator sequence and the *trp* promoter in front of the *bla* gene. In theory, this would put
the expression of the Beta-lactamase enzyme under the control of phosphate levels.

---

### Build

When building our constructs, we had to make sure all of the components we were trying to put
together were exactly right. The *phoA* promoter contains a *phoB* box that is necessary for the promoter to be
regulated properly so the complete sequence of the promoter including the *phoB* box and -10 and -35 sequences
needed to be accounted for. This had to be aligned properly with the transcription start site of the *trpR*
gene so the entire transcript would be transcribed and subsequently translated. The *trpL* promoter containing
the *trpL* operator sequence had to be determined and placed ahead of the *bla* gene. The *bla* gene also had the
*trpL* transcription start site, with the *trpL* ribosome binding site, ahead of the *bla* open reading frame.

We made two additional constructs that had *ssrA* degradation signal sequences attached to either
the *trpR* or the *bla* genes. This is a specific DNA sequence that is added to the 3’ end of the gene that
encodes a short peptide that leads to a shorter half-life of the protein. In both cases, the signal sequence
had to be place in the correct reading frame at the end of the gene but before the stop codon.

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### Test

After we had these constructs synthesized by Twist Biosciences, we transformed them into *E. coli*
and began to test them. Initially, we exposed the *E. coli* strains containing the constructs to high
concentrations of phosphate buffers to determine if we would get Beta-lactamase expression. We would
determine if Beta-lactamase was being expressed by whether the red color was being produced when nitrocefin
was added. We had a positive control that was a strain of *E. coli* with a plasmid that constitutively
expressed the *bla* gene so there was a high amount of Beta-lactamase present and a deep red color after
nitrocefin was added and allowed to sit for one hour. We also had a negative control which was a strain of
*E. coli* that did not contain the plasmid construct so no Beta-lactamase should be produced. In this case,
there was no significant red color observed when nitrocefin was added.

When exposed to high concentrations of phosphate buffer, we saw that the samples mostly turned
red after incubation with nitrocefin. The constructs that either did not have the *ssrA* degradation tag or
had the tag on the *trpR* gene produced a large amount of red color. The strain containing the construct with
the *ssrA* tag on the *bla* gene produced considerably less red color suggesting that the Beta-lactamase protein
was being degraded more rapidly so it could not cleave the nitrocefin as much.

After this experiment, we decided to expose the *E. coli* strains containing the constructs to
lower concentrations of phosphate that were more similar to what would be found in soil (approximately 30
ppm). We saw similar results as the first experiments with high phosphate concentrations except that the
strain containing the construct with the *ssrA* tag on the *trpR* gene was the only one that showed a positive
correlation between the concentration of phosphate and the amount of red color produced.

The last set of experiments we tried was to actually test soil samples for which we knew the
phosphate concentrations as determined previously through chemical tests. We only used the construct with
the *ssrA* tag on the *trpR* gene. While the results were not conclusive due to controls not working as well as
we would have hoped, we were able to show that we could see a difference between the phosphate levels in the
two soil samples that were similar to the known difference between the two. This suggested that with more
experimentation and fine tuning, it is possible to accurately measure bioavailable phosphate concentrations
using the system we developed.

---

### Learn

We learned that we need to make our plasmids as specific as possible to make the most efficient sensor. Understanding what can cause background noise and how we can limit it in our test samples was a consistent obstacle we had to troubleshoot throughout our experimentation. We have learned to be conscious of everything you use in your experiment, as even buffers can have major effects on your results. We learned a lot about the biology of operons and chemical cleaving through this project which our members can use in their future careers in biochemical research.

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## Overview:

This page gives the general protocols for several of the experiments we performed this year. If you would
like to see the results of these check out our [Results and Demonstration](https://2023.igem.wiki/wlc-milwaukee/results) page!

---

## Transformation:

Five constructs of plasmid were designed to be utilized as molecular biosensors for phosphate levels within
soil. All constructs were synthesized in the medium copy number plasmid “pTwist-kan”. Check out our [Parts](https://2023.igem.wiki/wlc-milwaukee/parts)
page to learn more about our constructs!

### Electroporation:

1. Labeled five microcentrifuge tubes with the appropriate plasmid
2. Transferred 50 µl DH5a electrocompetent *E. coli* cells to the microcentrifuge tube
3. Add 5 microliters of the plasmid to the corresponding microcentrifuge tube
4. Mix gently with the pipet tip
5. Remove the mixture of cells and ligation reaction from the microcentrifuge tube and add it to an
   electroporation cuvette making sure all the cells get to the bottom of the cuvette
6. Place the cuvette in the electroporator, Press and hold the button on the electroporator until a buzz is
   heard
7. After the buzz, remove the cuvette from the electroporator and add 1 mL LB broth to the electroporation
   cuvette quickly!
8. Remove the solution from the electroporation cuvette and place in a new clean microcentrifuge tube
9. Pipette the liquid (with cells) from the tube on an LB agar plate containing Kanamycin and spread the
   liquid on the plate with a plastic spreader
10. Place the plates in the 37˚ C incubator overnight

---

## Qualitative Color Change:

* Grew 5 ml liquid cultures overnight of the transformed *E. coli* strains. As well as two control strains.
  An *E. coli* which does not produce any Beta-lactamase as our negative control. An *E. coli* that produces a
  lot of Beta-lactamase independent of the phosphate concentration as our positive control.
* Made five 1:2 dilutions of 0.1 M KH2PO4 buffer
  + Four labeled microcentrifuge tubes had 0.5 ml water added to them
  + (0.1 M) The first (empty) tube had 1ml of the stock concentration of buffer added to it
  + (0.05 M) 500 µl from the first tube was added to the second tube and mixed by pipetting up and
    down
  + (0.025 M) 500 µl from the second tube was added to the third tube and mixed
  + (0.0125 M) 500 µl from the third was added to the fourth tube and mixed
  + (0.00625 M) 500 µl from the fourth was added to the fifth tube and mixed
* After the dilutions were prepared:
  + 500 µl of each culture was pipetted in new microcentrifuge tubes labeled the same as the diluted
    buffers.
  + 125 µl of each buffer dilution was added to each tube.
  + Approximately 5 minutes after the buffer was added, 4 microliters of 10 mg/ml Nitrocefin was
    added to all tubes
* Pictures were taken at 5 and 15 minutes after the addition of nitrocefin

---

## Soil Sample Preparation:

We tested two soil samples and two samples that came from biosolids.

* 2 grams of soil and biosolid samples were added to tubes
* 10 mM Tris pH 7.5 was added to the samples and vortexed for 30 seconds
* They were allowed it to incubate at room temperature for 10 minutes
* *E. coli* containing our functional construct and nitrocefin were then added to each sample and all samples were allowed to incubate for one hour
* After one hour, the samples were filtered through a 0.45 micron syringe filter which was attached to a 10 ml syringe and placed in a 15 ml conical tube
* Absorbance was measured on a spectrophotometer

---

## Spectrophotometry:

To analyze the minute color differences that we can't detect with the naked eye we used two methods of
Spectrophotometry.

### Light Spectrophotometry:

* Spectrophotometer ran at wavelength of 510 nm
* Absorbance readings were taken for the different dilutions described above

### UV-Vis Spectrophotometry:

* Absorbance readings were taken for the different dilutions described above.
* 1 ml of sample from each tube was transferred to a UV-Vis cuvette
* 2 mL of DI water was added to bring the sample to an appropriate volume
* Cuvettes containing these solutions were analyzed by the UV-Vis spectrophotometer at wavelengths from
  approximately 200 nm to 820 nm
* Readings from the spectrophotometer were transferred to a spreadsheet file and the data was analyzed

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## Project Achievements, Overview:

The goal of our project is to develop a simple but accurate test of bioavailable phosphate in
soil. We created three new constructs to measure bioavailable phosphate in soil using the Trp repressor as a
means to regulate the expression of Beta-lactamase. The amount of Trp repressor present would be regulated
by the *phoA* promoter which is regulated by the concentration of phosphate present. The *phoA* promoter is
activated when there is less phosphate present and turned off when there are higher amounts of phosphate
present. Since this *phoA* promoter is controlling the expression of *trpR*, there would be less negative
regulator controlling the expression of the *bla* gene (Beta-lactamase) when there is more phosphate present.
When the substrate of Beta-lactamase nitrocefin is present, a red color is produced. Therefore, the ideal
result in the construction of our constructs is to cause more red color to be produced when more phosphate
is presented to the *E. coli* containing the constructs and less red color when there is less phosphate.
Ultimately producing a quantifiable colorimetric nutrient concentration readout for inorganic/bioavailable
phosphate.

---

## Initial characterization of parts (Do our constructs work?)

After the constructs were designed, synthesized, and arrived from Twist Biosciences the first
experiment to perform was one to determine if they worked at all. This experiment is described on the
Experiment page. The appropriate solutions of bioavailable phosphate, Beta-lactamase substrate, and
nitrocefin were added to each sample and then waited to see if the solutions turned red.

![](https://static.igem.wiki/teams/4916/wiki/fall23/results1-6.png)

Figure 1 – Phosphate constructs with dilutions of sodium phosphate buffer

From left to right, (top row): Culture 1 with the different buffers. Culture 2 with the different
buffers. Culture 3 with the different buffers. (Bottom Row): Culture 4 with the different buffers.
Culture 5 with the different buffers. Culture that did not have a plasmid with the *bla* gene so no
Beta-lactamase was produced (negative control) with the different buffers.

The picture above shows one of the phosphate-inducible constructs. It was very apparent that
Beta-lactamase was being produced in most of our strains, although there was not a noticeable difference in
red color with the different phosphate concentrations.

We have performed a few repeats of the experiment and eventually all of our constructs have been
showing Beta-lactamase expression, but we haven’t been able to determine a quantitative correlation.

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.

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## Data analyzed with UV-Vis Spectrophotometry

Below is a procedure that was developed by Alyssa Ebeling last year. It was repeated for all
samples run on the UV-Vis.

1. The absorbances were plotted against the wavelength. The wavelengths were narrowed down to 300 to 700 nm
   to remove some of the deviation and focus on the target absorbance.
   * The height of the bump from the expected curve was the data point we would like to record as our
     absorbance.
![](https://static.igem.wiki/teams/4916/wiki/fall23/results7.png)
2. We then removed the wavelengths that made up the target bump.
3. An exponential function was graphed to find the baseline corrected absorbance.
![](https://static.igem.wiki/teams/4916/wiki/fall23/results8.png)
4. We used the peak of the graph of the baseline corrected absorbance as the absorbance for that specific
   concentration.
![](https://static.igem.wiki/teams/4916/wiki/fall23/results9.png)
5. After all the baseline corrections, all of the absorbances were plotted against concentration on the
   graph below.
![](https://static.igem.wiki/teams/4916/wiki/fall23/results10.png)

We had expected to see a negative correlation between the absorbance and concentration which is
not represented in this data. However, we did expect the negative control to be lower than all the
absorbances which are represented in this data.
This shows us that while our constructs are working, we still have not been able to make a standard curve.

## Light Spectrophotometry

Another set of experiments were performed and Light Spectrophotometry was utilized to analyze
the results.
The first construct is the *phoA* promoter controlling the *trpR* gene which codes for the TrpR protein. The
TrpR protein, when it interacts with tryptophan, binds to the *trpL* operator sequence to turn off the
expression of the *bla* gene. We performed an experiment where we grew *E. coli* containing this construct and
then added varying concentrations of phosphate buffer ranging from 0 to 113 ppm phosphate (Figure 1). This
experiment was performed in triplicate and the points on the graph are the averages of the three results.

![](https://static.igem.wiki/teams/4916/wiki/fall23/results11.png)

Unfortunately, the results show an opposite trend than what we expected. We were hoping to see
an increase in OD510 (red color) as phosphate concentration increased which would suggest that there is more
Beta-lactase present when phosphate concentrations increase. Possible reasons for this discrepancy between
our results and our expectations is that there is a high amount of background in the experiments. Even when
no phosphate is added, there is a relatively large amount of red color in the samples. This suggests there
is Beta-lactamase present in the cells even when no phosphate is added. We performed control experiments to
show that when there is no *bla* gene in the *E. coli*, there is no red color produced so the red color is
dependent on the expression of *bla*. There is also no *bla* gene in our *E. coli* strain except for what is
provided in our construct on the plasmid. This tells us that there is expression of the *bla* gene in our
construct independent of the phosphate-inducible expression we are trying to perform.
In order to address the issue of high background in the samples we created two other constructs. They are
both similar to the first except that the new constructs have a protein degradation tag incorporated on
either the *trpR* gene or the *bla* gene. The first of these new constructs had an *ssrA* DNA sequence added to
the 3’ end of the *trpR* gene which should cause the TrpR protein to be degraded more quickly after it is
translated. The idea behind this is that background TrpR that may have been produced before an experiment
would be degraded more quickly which would allow for a greater sensitivity to new TrpR being produced during
the experimental conditions. To test this, we performed an experiment similar to the experiment in Figure 1
to see if this construct with the degradation tag on TrpR would give us results where increased phosphate
concentration correlated with an increase in red color.

![](https://static.igem.wiki/teams/4916/wiki/fall23/results12.png)

In Figure 2, we see an increase in OD510 (red color) when phosphate concentration increases
which is what was expected. Also, in this graph, we attempted to account for the background red color by
subtracting the OD reading for the sample that did not receive any phosphate from all of the other samples.
The line produced by the linear regression had an equation of y=0.0006x + 0.0432 which we could use to
determine the concentration of phosphate in a dirt sample after exposing the dirt to *E. coli* containing the
construct and nitrocefin.
The third construct that we made was the same as the first except that it had the *ssrA* degradation tag
attached to the *bla* gene. Again, the goal of this was to reduce background color. If Beta-lactamase was
being produced in the cells prior to the experiment, the degradation tag could reduce how long the enzyme
remained active by degrading it more quickly. When *E. coli* containing this construct were mixed with the
various phosphate concentrations, the overall amount of red color was reduced in these cells but the trend
line showed a decrease in red color as phosphate levels increased similar to what was seen with the first
construct (Figure 3). This demonstrated that this construct likely was also not going to be useful for
measuring bioavailable phosphate concentrations in the soil.

![](https://static.igem.wiki/teams/4916/wiki/fall23/results13.png)

Given these results, we found a construct (Figure 2) that appeared to give us increased red color
with increased phosphate concentrations using phosphate buffers prepared in the lab.

---

## Soil test

Our next goal was to determine if we could determine phosphate concentrations in soil. We tested
two soil samples and two samples that came from biosolids. Biosolids are treated byproducts of waste water
treatment plants than can be used as amendments to fields. To test these samples, we weighed two grams of
each dried soil or biosolid sample and added it to 10 mM Tris buffer and allowed it to incubate at room
temperature for 10 minutes. *E. coli* containing our functional construct and nitrocefin were then added to
each sample and all samples were allowed to incubate for one hour. After one hour, the samples were filtered
through 0.45 micron syringe filters and absorbance was measured on a spectrophotometer. Unfortunately, the
biosolid samples did not filter as cleanly as the soil samples as there was significant brown coloration or
cloudiness after filtration. Thus, we did not try to calculate phosphate concentrations with these samples.
The soil samples provided samples that could be measured so we continued calculations with these.
While we performed the experiments with soil and biosolid samples, we also performed control experiments
with the phosphate buffers again except mostly following the conditions we used to test the soil and
biosolid samples. We did not filter the samples since there were no solids. Unfortunately, the graph that
resulted (Figure 4) from the control experiments with the phosphate buffers produced a negative correlation
similar to the original construct (Figure 1) and the construct with *ssrA*-tagged *bla* gene (Figure 3).

![](https://static.igem.wiki/teams/4916/wiki/fall23/results14.png)

It is possible that the greater volumes (roughly 10 times greater) we were using in the
experiments involving the soil samples affected the experiment so it would be worth going back and trying
the control experiments with all of the constructs in these conditions to see if they all reacted similarly.
However, if we took the absorbance values from the soil experiments and calculated phosphate concentrations
using the equation determined for the linear regression line in Figure 2, we would get the values we see in
Table 1 below.

![](https://static.igem.wiki/teams/4916/wiki/fall23/results15.png)

The values we determined for phosphate concentration using the *E. coli* test that we developed
were very close to the known values determined previously using chemical tests. However, we cannot claim
this as completely accurate since we are using the standard curve from one experiment with the results from
another. However, if time permitted we would certainly like to repeat the soil and control experiments to
see if we could get valid control and sample results that would allow us to make sound scientific
conclusions. That being said, it is encouraging that the results in this table are somewhere in the range of
previously known results and that they have a similar ratio relative to each other. This suggests that
future experiments could demonstrate that our *E. coli* testing method is a viable way of testing soil for
bioavailable phosphate concentrations.

---

## Future work

Last year we strived to test multiple nutrient sensors and we had said we would have liked to
test more nutrient types. This year we have modified the phosphate contrasts we had made last year and got
more promising results. We were able to demonstrate that Beta-lactamase is being produced from the construct
when exposed to phosphate. However, more experiments need to be performed to fine-tune the amount of
Beta-lactamase being produced to create a standard curve so we can use our sensor to provide an analytical
output.

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