Results

Defluorinating Enzyme Library testing

---

Optogenetic Control Backbone:

To facilitate the expression and cloning of our defluorinating enzymes, we decided to set up an standardized Golden Gate template

Therefore, we could proceed forward with a backbone that easily distinguished between positive pcr reaction results and negative results; we did not need to independently resequence our plasmid for each set of reactions. To aid future iGEM teams in their Golden Gate reactions, this backbone has been published as parts BBa_K4895203, BBa_K4895202, and BBa_K4895201.

Initial Defluorinating Enzyme Assays

To test initial enzyme activity, we plated bacteria containing defluorinating enzymes onto a 2,2-Difluorohexanoic acid assay.

The above results suggest that one of many things could be occuring:

1. The concentration of difluorohexanoic is too low to pose a threat to the bacteria, but it is high enough to keep selective pressure on the plasmid
2. The enzymes or even the plasmid is toxic to our bacteria, which confers an advantage to WT
3. The particular fluorinated compound we are using is not toxic enough to our bacteria

From here, we decided to further investigate by performing liquid cultures with Kanamycin resistance from the growth on the plates. We then performed an assay of colony PCRs, which all came back positive. Although there was not a definitive conferred benefit of our defluorinating enzymes, it seemed that the bacteria that had the defluorinating enzymes were under selective pressure. Therefore, this is a success, as we proved that, without Kanamycin antibiotic selection, even as little as 1mM of saponified 2,2-Difluorohexanoic acid is enough selective pressure to keep defluorinating enzymes.

If we are to continue with this experiment, there would be more research done into the application of the fluorinated compounds within our media, to include potentially adding it to cooled agar, or working with solely liquid cultures. Furthermore, there could be more literature review into the exact mechanism of PFAS accumulation in cells.

Ectopic Metabolic Pathway

---

In order to further investigate the initial difference in growth, we set up a multivariate assay to determine the relationship between:

1. Bacteria augmented with our Ectopic Metabolic Pathway,
2. Aerobic Growth,
3. Anaerobic Growth,
4. and Wild-type Bacteria.

Setup and experimental methods can be found here

Data Collection

To better facilitate and automate data collection, we utilized software tools and augmented media. To increase contrast and visibility of growth on media, we used Trypan Blue in our M9 media. The software tool we chose, imageJ, is able to detect light filtering levels through the plate. So, theoretically, more growth on the plates would result in less blue light filtering through, thus giving us easily quantifiable and collectable growth data points. In practice, we set up a series of 4 controls, which contained no bacteria so therefore they should set a baseline for light filtering levels. A special feature of Trypan Blue is that living bacteria will not be stained with the dye, and this further bolsters the automation of data collection. The control plates represent the isolated blue channel, and set a baseline for the mean amount of blue light filtration on plates with no growth. The software then directly exports the data as an array. Then, a series of statistical tests were performed.

Statistical Analysis

A Google Collab book was used to conduct the statistical analysis and to generate figures. The data was imported with pandas and an ANOVA was done using pingouin. A pairwise Tukey's test was then performed to do a comparison for the group*colony*treatment. No significant differences were found from the Tukey's test between any G4 replicate and WT (lowest p-value = 0.398).

Our ANOVA returned the below table

Source	Sum of Squares	Degrees of Freedom	Mean Squares	F-statistic	P-score
Group(G4 vs WT)	-2.64e-13	1	-2.65e-13	-4.26e-11	1
Colony	-2.91e-16	5	-5.83e-17	-9.41e-15	1
Treatment(Aerobic vs Anaerobic)	1.83e-16	2	9.15e-17	1.48e-14	1
Group*Colony	1.71	5	0.3438e	55.52	<0.0001
Group*Treatment	1.992e-4	2	9.958e-5	1.608e-2	0.984
Colony*Treatment	3.998e-4	10	3.998e-5	6.457e-3	0.9999
Group*Colony*Treatment	2.718e-1	10	2.718e-2	4.39	0.0003
Residuals	2.539e-1	41	6.192e-3	NA	NA

After further reviewing the data to ensure that nothing was missed, a violin plot was used to visualise the potential for skew. After seeing the violin plot, it was determined that we should evaluate the Pearson coefficient of skew and test for skewness. This returned a coefficient of 1.04, which is inconclusive, but suggests that there is not much skew to the data. Thus, it can be seen that there is not much of a difference between the datasets for WT and augmented Bacteria. However, because some skew is present, we decided to use a Kruskel-Wallis H-test. This would avoid colony variance, and focus on the larger group identity of WT and augmented Bacteria.

This returned a P-value that indicated that our medians are from the same population. This means that there is no statistically significant difference between the two data sets, and we cannot conclude that there is any difference between WT and augmented bacteria populations.

To determine the likelihood of a type II error, a power test was conducted revealing a beta of 0.129. For future experiments it was determined that the proper sample size to obtain a power of .80, would need to have n = 124. In future assay attempts, we will be sure to use larger datasets to lessen the variance of our data as well as better understand the variables at play.

As a result of this inconclusive data, we determined that simply adding in the metabolic enzymes would not nearly be enough; to confer complete reliance on our ectopic metabolic pathway, we would need to knock out endogenous metabolic enzymes. In the future, we will revisit this aspect of the project once we can confirm the complete knockout of associated metabolic enzymes.

MiKOP: Micro Knockout Plasmid

In order to determine the success of our transformation we performed two separate assays:

1. Liquid Cultures: From the antibiotic plate, 8 colonies are picked and placed in 4mL of antibiotic resistance LB media. In a separate culture tube, WT cells are placed into antibiotic resistance LB media as well. Separate colonies on the plates were placed in Kanamycin media, for a negative control
2. Colony PCR: The 8 colonies that were placed into antibiotic resistance media were also colony PCR'd, with a set of primers that would confirm the recombination event as well as expression of the plasmid.

With this qualitative assay, and the limited time left in the project, we were determined to get shareable results out in time.