iGEM 2019 — Team Waterloo

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# Team:Waterloo/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


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# Team:Waterloo/Experiments

iGEM UWaterloo 2019 - Experiments

[Experiments](https://2019.igem.org/Team%3AWaterloo)

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# Experiments

# Plant Inoculation, Growth, and Nodulation

## Seed Inoculation (*optional*)

1. Grow 3-4 YM plate lawns of *Bradyrhizobium diazoefficiens*.

   * The manitol in the YM media should allow for the bacteria to adhere better to the seed coat, and acts as a carbon source for them.
2. After lawn is grown up, asceptically add 10 mL of YM liquid media and scrape off culture (without breaking the agar), and add to a sterile beaker.
3. Repeat this process, adding another 10 mL to the plate, and wash off any extra culture that might remain. Add this to the previous beaker. You should have approximately 20 mL per plate from this.
4. Repeat for each plate.

   * Place all the liquid into a single beaker.
5. Soak 64 sterilized soybean seeds in this liquid culture for an hour, in the beaker.
6. Remove seeds from solution by covering another beaker with a cheesecloth or gauze, and pouring out the beaker into it (all seeds should be caught on the fabric).
7. Use hands or tweezers to remove the seeds - immediately plant into soaked vermiculite.

## Flood Inoculation (*optional*)

1. Grow 3-4 YM plate lawns of *Bradyrhizobium diazoefficiens*.
2. After lawn is grown up, asceptically add 10 mL of the bacteriological saline solution and scrape of off the culture from the plate, and add to a sterile beaker.
3. Repeat this process, adding another 10 mL to the plate, and wash off any extra culture on the plate.

   * Add this to the previous beaker used.
   * You should have approximately 20 mL per plate from this.
4. Repeat for each plate.
5. Use a sterile large syringe (60 ml) and add droplets (~ 5mL) to the root area of all the seedlings.
6. Try to concentrate this to directly above the roots.

   * Does not need to be sterile.
7. Place seedlings back into the incubator.

## Seed Sterilization

1. Place seeds in 50mL falcon tube.
2. Immerse in 0.1% Trixton X-100 for 2 minutes.
3. Decant into a waste beaker.
4. Immerse in 70% ethanol for 2 minutes.
5. Decant into waste beaker.
6. Rinse 3 times with DI H2O.

## Germinating and Planting the Seeds

1. After seed sterilization, wrap moist seeds on a paper towel and place in a plastic bag (a ziplock, for example) and leave for two days,

   * Check the seeds after the first 24 hours, and observe if germination has taken place.
2. After 2 days, or once you see sprouting from the seeds, plant in prepared pots.

   * (*To prepare planting*) Fill each pot with vermiculite, almost to the top.
3. Water the vermiculite with warm water, the aim here is to soak it until it becomes x5-x7 times heavier than it was before.

   * This is not-quantitative, and does not need to be precisely measured.
   * Ensure that: the water is warm, and that you don't oversoak.
4. Lightly water the pots with cold water, so that the top of the vermiculite is luke-warm to the touch.

   * Try not to oversoak the vermiculite.
5. Push three holes into the vermiculate that are one inch deep and equidistant from each other.

   * You can use a pencil.
   * Each hole should be 1/2 inch - 1 inch in depth. The point of using the pencil is that the sharpened part of the pencil should be about that long. The holes don't have to be perfectly separate, but just far apart enough that if each seed added to the hole was to germinate, the root systems wouldn't entangle.
6. Add 3 seeds per pot, place in the incubator and wait 5 days.

   * Add one seed to each hole.

## Care for Plants and Bacterial Treatment

* (*optional*) after shoots show, flood with bacterial inoculum.
* Remove any underdeveloped shoots, always leaving one growing plant in the pots.
* If the vermiculite is moist to the touch, even after two days, do not re-water.
* Immedietly water if the vermiculite is dry, even if the two days haven't passed.

### To water:

1. The plants should be watered once the top of the vermiculite is drier to the touch, on the top.
   * Water with DI water.
2. Fill a watering can and topically apply to the plants.

# Minimum Inhibitory Concentration (MIC): Herbicide Tolerance Assays

## MIC assay design

We would like to thank [Newcastle iGEM](https://2019.igem.org/Team%3ANewcastle/Collaborations#UofW "Waterloo 2019 Collaborations"), and specifically Connor, for their support in this area of the project.

MIC assays are used in many areas of microbiology, with applications including evaluation of novel antimicrobial agents (Foerster, 2016). Organisms generally grow faster at lower concentrations of a given toxin, giving a profile as shown in figure 1. The profile’s shape is explained as follows: at concentrations below a critical toxin concentration, the organism is completely uninhibited, allowing it to grow at a maximal rate. At concentration above a critical toxin concentration, the organism is mostly inhibited, resulting in minimal growth. At the critical “minimum inhibitory” concentration, a much steeper transition region exists. The position and steepness of this transition region reflect toxicity: an organism with a higher toxin tolerance

![Fig1](https://static.igem.org/mediawiki/2019/f/f2/T--Waterloo--Fig1.png "Profile of Microbial Growth Rate Versus Toxin Concentration")

Figure 1 above represents an idealized profile of microbial growth rate versus toxin concentration.

Various MIC formats and methods exist, but we chose to use a 96-well plate format, allowing many toxin concentrations to be evaluated in replicate at the same time. A plate-reading spectrophotometer was then used to measure the bacterial growth in each well using absorbance of light at 600 nm (Abs600).

We conducted MIC experiments with both *E. coli* and *B. diazoeffiens* and evaluated tolerance toward both linuron and 3,4-DCA.

We used a combination of approaches to evaluate growth, measuring Abs600 at a single timepoint for some experiments and Abs600 over time for other experiments to calculate growth rate. Single-timepoint MICs are much more convenient to perform, and show a snapshot of growth represented as Abs600 vs concentration. This is sufficient for some purposes, but fails to take into account the overall growth characteristics of a each bacterial population, especially when different strains or species are being compared. For example, two bacterial strains may have the same growth rate during exponential phase, but if one had a slightly longer lag phase, its Abs600 will appear lower at a given point in time, falsely suggesting poorer toxin tolerance. Single timepoint OD values are also affected greatly by small differences in the OD of the added broth culture. Collecting Abs600 data at several timepoints allows for calculation of growth rate, a much more robust measure of a strain’s ability to grow at a certain toxin concentration.

Timepoints for measuring growth were determined by trial and error, with the goal of taking measurements during mid-log phase. This phase of growth corresponds to the greatest differences between bacterial populations growing at different rates for single readings and provides the best estimation of growth rate if multiple readings are used. For *E. coli*, single timepoints were taken at 7 hours. For *B. diazoefficiens*, single timepoints were taken at 24 hours and growth curves from 22-30 hours.

In summary, our MIC protocol involved the following:

* Grow organism of interest in appropriate liquid media to mid-exponential phase, verifying by measuring Abs600
* Prepare a solution of the toxin of interest in the same growth media used for the liquid culture
* Prepare serial dilutions of the toxin of interest across the columns of 96-well plate
* Add a fixed volume of the mid-exponential phase liquid culture to each well
* Incubate the 96-well plate, allowing the organism to grow
* Read Abs600 of each well at one or more timepoints to evaluate growth

This is presented in greater detail in the below protocol.

[[Link to protocol]](https://static.igem.org/mediawiki/2019/9/98/T--Waterloo--MICProtocol.pdf)

Due to *E. coli\*’s fast growth rate, we chose to dilute it from overnight culture in LB broth to a target Abs600 of 0.1 before each experiment. \*B. diazoefficiens* was allowed to grow for approximately 4 days and 16 hours to an Abs600 of 0.4-0.6, which is thought to correspond to mid-exponential phase.

Since linuron and 3,4-DCA both have limited water solubility, dissolving directly into LB or TY media was not practical, even at concentrations well below the solubility. Instead, concentrated solutions of linuron and 3,4-DCA were prepared in DMSO (approx. 45 mM and 90 mM respectively), and these were then used to prepare working solutions of 290 µM linuron and 550 µM 3,4-DCA. The very low concentration of DMSO in the solutions was assumed to be of negligible toxicity to the organisms.

The 96-well plate’s border was filled with plain LB or TY broth in order to serve as a barrier to evaporation during incubation and to act as a control for potential contamination.

## Analysis of MIC data

For single-timepoint readings, Abs600 data were corrected by subtracting the average broth-only blank.

For growth curve experiments, Abs600 data were plotted versus measurement time in hours and fit with a curve of the form y = aekx + b, where k is growth rate in reciprocal hours.

Abs600 for growth rate was analysed by plotting versus toxin concentration. A horizontal log scale was used for convenience. The data were then fit using an empirically-chosen 4-parameter sigmoidal curve of the form shown in equation 1 (Tixier, 2002).

$$ y=A+\frac{B}{1+\exp(C(\ln D - \ln x))} $$

This was completed using least squares regression in Microsoft Excel Solver. The four parameters serve to summarize the relationship between growth rate or Abs600 and pesticide concentration. This relationship also allows the calculation of parameters such as inhibitory concentrations, such as IC50, IC10, or IC90, which represent the amounts of toxin required to toxin concentrations required to inhibit 10, 50, or 90 % of growth respectively. Inhibitory concentration at a given level f is calculated from the sigmoidal fit parameters as follows: yf = A + B(1 – f), the IC50 is calculated by substituting yf into the four parameter fit above and solving for x, which corresponds to ICf.

![Fig2](https://static.igem.org/mediawiki/2019/1/17/T--Waterloo--Fig2.png)

Figure 2 above is an idealized representation of IC10, and IC90.

### Waterloo iGEM 2019

"Waterloo iGEM: hacking synbio since 2006"

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# Team:Waterloo/Results

iGEM UWaterloo 2019 - Results

[Results](https://2019.igem.org/Team%3AWaterloo)

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* [Description](https://2019.igem.org/Team%3AWaterloo/Description)
* [Design](https://2019.igem.org/Team%3AWaterloo/Design)
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# Results

# Cloning

## Linuron construct

![Linuron](https://static.igem.org/mediawiki/2019/d/d2/T--Waterloo--Linuron.png)

In this construct, the insert is was cloned into the BglII site between the Paph promoter and bjGFP sequence on the pRJPaph-bjGFP to give a transcriptional fusion with the linuron degradation genes. The insert itself was made by inserting the BBa\_B0034 ribosome binding site ahead of each the *libA* and *nat1* genes through PCR. These were then inserted downstream of the Paph promoter in the order of *libA* then *nat1* by using Gibson Assembly/NEBuild. The coding sequences for the *libA* and *nat1* genes were codon optimised for *B. diazoefficiens* USDA 110.5.

We successfully assembled this construct! It was validated by colony PCR (amplifying inserted genes) and sequencing. We also confirmed operon expression by measuring GFP fluorescence.

## Glyphosate construct

![glyphosate](https://static.igem.org/mediawiki/2019/a/a4/T--Waterloo--Glyphosate.png)

Similar to the construction of our plasmid for resistance to linuron, we planned to insert the BBa\_B0034 ribosome binding site and coding sequence of the CP4 EPSP synthase gene into the BglII site present between the Paph promoter and bjGFP coding sequence on the pRJPaph-bjGFP plasmid to give a transcriptional fusion of our EPSP gene with the bjGFP. The coding sequence of the CP4 EPSP synthase gene used was derived from the amino acid sequence claimed in the Monsanto patent, reverse translated to be codon optimised for *B. diazoefficiens* USDA 110.5 Attempts to assemble this construct through NEBuilder were not successful as the no conditions could be found to amplify the coding sequence with required overhangs and ribosome binding site by PCR. This likely was due to the high GC content of the coding sequence (71.2%), however the lengths of the primers could have also contributed to the difficulty. Unfortunately, we decided to drop the glyphosate part of the project and only focus on Linuron due to time constraints.

# MIC assay results

## Growth curve experiment with wild-type *B. diazoefficiens*

Although we were unable to evaluate the tolerance of engineered *B. diazoefficiens* strains due to time constraints, we nonetheless investigated the tolerance of wild-type *B. diazoefficiens* to linuron and 3,4-Dichloroaniline (3,4-DCA). This was evaluated using growth curve Minimum Inhibitory Concentration (MIC) experiments performed with hourly absorbance readings from 20 to 25 hours of incubation at 30 °C. The absorbances at the five timepoints were plotted versus time and fit with exponential curves of the form y = ae^(kx) + b, where k is growth rate. Sextuplicates were used for each toxin and concentration.

The results of these experiments are presented in figures 5, 6, and 7.

![LinBd2](https://static.igem.org/mediawiki/2019/6/6d/T--Waterloo--LinBd2.png)

*Figure 5*. Growth rate versus linuron concentration from 20 to 25 hours. Error bars represent standard deviations of sextuplicates. The growth rate with no toxin was 0.08639 +/- 0.01232 1/h. The sigmoidal fit parameters A, B, C, and D are 3.23E-03, 3.46E-02, 2.96, and 2.30E+01 respectively.

![DCABd2](https://static.igem.org/mediawiki/2019/0/06/T--Waterloo--DCABd2.png)

*Figure 6*. Growth rate versus linuron concentration from 20 to 25 hours. Error bars represent standard deviations of sextuplicates. The growth rate with no toxin was 0.08639 +/- 0.01232 1/h. The sigmoidal fit parameters A, B, C, and D are 7.43E-03, 3.62E-02, 8.32E-01, and 2.08E+01 respectively.

*Figure 7*. Summary of key inhibitory concentrations for linuron and 3,4-DCA with *B. diazoefficiens* based on growth curves from 20 to 25 hours.

![T1](https://static.igem.org/mediawiki/2019/8/83/T--Waterloo--T1.png)

These differences in toxicity profiles are demonstrated by the key inhibitory concentrations in figure 7. Linuron’s IC50 value is higher than that of 3,4-DCA, demonstrating that linuron is generally less toxic than 3,4-DCA. This agrees with reported toxicity values (Tixier, 2002) (Santos, 2014). Comparing IC10 values shows that 3,4-DCA starts showing toxic effects at a much lower concentration than linuron, due to steeper slope of the plot for linuron. The steeper slope of linuron’s plot also means that linuron’s IC90 is much lower than that of 3,4-DCA. This means that full growth inhibition actually requires a lower concentration of linuron than of 3,4-DCA, despite linuron’s lower IC50. These differences highlight the importance of examining the entire toxicity profile and various inhibitory concentrations, since these convey much more information that the IC50 value alone.

We have therefore experimentally deteremined the toxicity profiles of both linuron and 3,4-DCA toward wild type *B. diazoefficiens*, validating our experimental approach. This information provides also a reference point against which to compare engineered strains, other toxins, or bacterial species.

## Single-timepoint experiment with wild-type *B. diazoefficiens*

In addition to the growth curve experiment peformed with *B. diazoefficiens* both in linuron and 3,4-DCA, we also completed this experiment in single-time point format. This was done using one Abs600 after 22 hours incubation at 30 C. Figures 8 and 9 show plots of Abs600 versus toxin concentration. These results are shown in figures 8, 9, and 10. Fitting was performed to the four parameter in equation 1.

![LinBd1](https://static.igem.org/mediawiki/2019/5/5b/T--Waterloo--LinBd1.png)

*Figure 8*. Abs600 versus linuron concentration at 22 hours. Error bars represent standard deviations of sextuplicates. The sigmoidal fit parameters A, B, C, and D are 1.28E-02, 5.52E-02, 1.62, and 1.28E+01 respectively.

![DCABd1](https://static.igem.org/mediawiki/2019/e/ed/T--Waterloo--DCABd1.png)

*Figure 9*. Abs600 versus linuron concentration at 22 hours. Error bars represent standard deviations of sextuplicates. The sigmoidal fit parameters A, B, C, and D are 1.50E-02, 5.47E-02, 8.97E-01, and 3.08E+01 respectively.

*Figure 10*. Summary of key inhibitory concentrations for linuron and 3,4-DCA with *B. diazoefficiens* after 22 hours.

![T2](https://static.igem.org/mediawiki/2019/2/2d/T--Waterloo--T2.png)

Figures 8 and 9 both show the characteristic sigmoidal shapes expected for MIC experiments. All datapoints are very close to the sigmoidal fit, indicating that this model is a good representation of the data.

Figure 10 summarizes some key inhibitory concentrations as calculated based on these growth curves. Although the two curves have the same general shape as those determined using the growth curve MICs, the calculated inbitory concentrations are somewhat different. The single timepoint data’s IC50 values suggest that linuron is more toxic that 3,4-DCA, which disagrees with the results obtained using growth curves and literature toxicity data (Tixier, 2002; Santos, 2014). This highlights the shortfalls of determining toxicity from Abs600 at a single timepoint and the importance of using growth curves to evaluate a toxin’s overall effect on an organism’s growth rate.

## Single-timepoint experiment with *E. coli* empty vector and constructs

We also chose to evaluate the linuron and 3,4-DCA tolerance of our engineered DH5α strains. We performed single-timepoint MICs after 7 hours incubation at 30 C using empty vector pRJPaph-GFP and two constructs, assigned the numerical values of 14 and 21. These constructs had been vertified by colony PCR, sequencing, and GFP fluorescence reading. Each single-timepoint MIC was completed in triplicate, using LB supplemented with tetracycline to promote vector retention. Figures 3 and 4 show plots of Abs600 versus log toxin concentration for each.

![LinEc](https://static.igem.org/mediawiki/2019/c/c9/T--Waterloo--LinEc.png)

*Figure 3*. Abs600 versus linuron concentration for 3 engineered DH5α strains at 7 hours. “EV” is pRJPaph-GFP and “14” and “21” each contain the construct with LibA and NAT1. Datapoints are averages of triplicates.

![DCAEc](https://static.igem.org/mediawiki/2019/a/a4/T--Waterloo--DCAEc.png)

Figure 4. Abs600 versus 3,4-DCA concentration for 3 engineered DH5α strains at 7 hours. “EV” is pRJPaph-GFP and “14” and “21” each contain the construct with LibA and NAT1. Datapoints are averages of triplicates.

Linuron (figure 3) does not show any significant inhibition, even at the highest linuron concentrations. This means that it is not possible to compute parameters such as IC50, so sigmoidal fitting was not applied. Since higher linuron concentrations would not be possible due to solubility constraints, this type of assay is a poor method for evaluating LibA and NAT1 activity in *E. coli*. Additionally, there are no significant differences between the Abs600 values of the different strains, suggesting that the construct and empty vector do not cause measurable differences in growth rate.

3,4-DCA (figure 4) shows nearly equal inhibition of all three strains beginning at 226 µM 3,4-DCA. However, since the lower “minimal growth rate” region is absent, sigmoidal fitting was not applied, so it is not possible to calculate paramters such as IC50. As with linuron, obtained data at higher toxin concentrations would be challenging due to solubility constraints. Additionally, there are no significant differences between the Abs600 values of the different strains at any toxin concentration, suggesting that the construct and empty vector do not cause differences in growth.

These experiments therefore demonstrate that the construct does not lend linuron or 3,4-DCA tolerance to *E. coli* DH5α. However, the lack of difference in growth rate provides some evidence that LibA and NAT1 do not impose a significant metabolic burden on DH5α when used in this expression system.

# References

Santos, S., Romeu, V., Maria, F., Sharon, C., António, M., Joaqium, V., & Amália, J. (2014). Toxicity of the herbicide linuron as assessed by bacterial and mitochondrial model systems. Toxicology in Vitro, 28 doi:10.1016/j.tiv.2014.04.004

Tixier, C., Sancelme, M., Aït-Aïssa, W., P., Bonneymoy, F., Cuer, A., Fruffaut, N., & Veschambre, H. (2002). Biotransformation of phenylurea herbicides by a soil bacterial strain, arthrobacter sp. N2: Structure, ecotoxicity and fate of diuron metabolite with soil fungi. Chemosphere, 46, 519-526. doi:10.1016/S0045-6535(01)00193-X

### Waterloo iGEM 2019

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