iGEM 2022 — Team wvhs-sandiego

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{% block title %}**Notebook**{% endblock %}
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## Summary

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All lab work completed was done at the J. Craig Venter Institute under the supervision of Dr. Erin Garza and Dr. Jocelyn Macho. The following page includes all experiments conducted in lab as well as a brief overview of their purposes. Our assembly methods were loop assembly that utilized both Golden Gate and Gibson Assembly to combine our pL0 and pL1 parts.

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{% block title %}**Engineering Success**{% endblock %}
{% block lead %}We went through multiple iterations of the engineering design cycle{% endblock %}
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{% block title %}**Experiments**{% endblock %}
{% block lead %}Research, experiments, and protocols used in our iGEM project.{% endblock %}
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## Summary

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All lab work completed was done at the J. Craig Venter Institute under the supervision of Dr. Erin Garza and Dr. Jocelyn Macho. The following page includes all experiments conducted in lab as well as a brief overview of their purposes. Our assembly methods were loop assembly that utilized both Golden Gate and Gibson Assembly to combine our pL0 and pL1 parts.

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{% block title %}**Results**{% endblock %}
{% block lead %}Analyzing our Data{% endblock %}
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## Results

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The Defluorinator project was created to remove PFOA from water systems. This can be done by breaking the carbon fluorine bond in PFOA which will cause the chemical to break down. Two enzymes haloacid and fluoroacetate dehalogenase were chosen to break the fluorine bonds in PFOA. Plasmid was put into E. coli.

## Testing for PFOA

To determine if this sample was capable of defluorinating PFOA we measured the pH and optical density in 6 different cultures: E.Coli with engineered FAcD + PFOA, E. coli with engineered HAD + PFOA, NEB5a E.coli + PFOA, and wild type P. putida + PFOA without plasmid. With two controls of engineered FAcD in e.coli, engineered HAD in E.coli, and just LB broth. pH level and optical density were measured at several time points over 3 days or 54 hours (12:00 PM, 3:00 PM, 6:00 PM, 9:00 AM, 12:00 PM, 3:00 PM, 6:00 PM, 9:00 AM, 12:00 PM, 3:00 PM, 6:00 PM).

![](https://static.igem.wiki/teams/4191/wiki/culture-key-results.png)

![](https://static.igem.wiki/teams/4191/wiki/screen-shot-2022-10-11-at-4-41-40-pm.png)

Figure 1 displays the data found for pH vs. time.

pH testing was done to determine the amount of fluorine ions that were present. As PFOA was degraded, there is a byproduct of free floating hydrogen and fluorine ions. The fluoride ions will bond with free hydrogen ions creating hydrofluoric acid (HF), an acidic compound. The more HF that is being created, the more H+ in solutions we will be measuring, which means a lower pH level. The less free H+ in a system, the more basic it would be. As pH decreases, the amount of PFOA decreases.

![](https://static.igem.wiki/teams/4191/wiki/screen-shot-2022-10-11-at-4-42-18-pm.png)

Figure 2 displays the data found for optical density vs. time.

The graphs of pH vs. time for FAcD + PFOA, HAD + PFOA, NEB5a e coli + PFOA shows generally a steady decline from 5 to 25 hours. In order to check if our experiment produced the desired results due to the engineered enzymes, we compared the effects of our bacteria to the effects of the wildtype strain. The pH of wild type P. Putida increases from 6.5 to 7.43 over the 54 hours and does not decrease. However, in our engineered FAcD the pH decreases from 6.39 to 5.83 and decreases from 6.37 to 5.91 in the engineered HAD. All cultures were put into LB broth and the control of just LB broth has a relatively stable pH of 6.5 throughout the 54 hours.

## Measuring Bacteria Growth

![](https://static.igem.wiki/teams/4191/wiki/ph-vs-time-in-hours-for-cultures.png)

Figure 3 portrays the data found for pH vs. time across the cultures.

Measuring optical density was used to determine how efficiently the bacteria was growing in the presence of PFOA. The optical density will increase when there is more bacteria present in the solution.

![](https://static.igem.wiki/teams/4191/wiki/optical-density-o-d-vs-time-hours-for-cultures.png)

Figure 4 depicts the data found for optical density vs. time.

There is a strong positive linear correlation between optical density and time for all the cultures. As time increased, the optical density also increased. This means that both HAD and FAcD were able to grow over the 54 hours.

## Errors

Our results also showed that after 26 hours the pH generally started to increase which could be due to several reasons. The gas form of HF produced could have left our system when opening the culture lids to test pH. pH might not have been the most efficient way to measure the degradation of data. The increase in pH after about 20 hours could also be due to all the PFOA being degraded, producing no more H+ ions. The degradation process could have also ceased to continue because our plasmid might not have been 100% efficient in producing these enzymes needed to break down 100% of the PFOA. Another reason could be that the bacteria itself produces basic metabolites to adjust to the environment, causing the solution to become more basic. This means that we cannot compare if the engineered HAD or FAcD degraded more PFOA.

## Future Research

To further investigate the degradation process of PFOA by the engineered HAD and FAcD, we would recommend using a different method for PFOA detection. Our experiments used pH because it was the most available option to our lab at that time. GC-Ms or LC-Ms methods could be used to determine how PFOA was degraded. If pH testing was to be used again, a closed system should be used so no HF gas escapes, increasing the pH.

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