iGEM 2021 — Team USAFA

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# Team:USAFA/Notebook

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# Lab Notebook

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# Team:USAFA/Engineering

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![](https://static.igem.org/mediawiki/2021/8/8d/T--USAFA--Engineering_Process.png)
 There were several iterations of the engineering process before the Defluorine Machine became a novel, functional, bioremediation tool. This process began with dehalogenase enzymes identified courtesy of last year's USAFA iGEM team. Dehalogenases are a class of hydrolase enzymes that specifically target carbon-halide bonds, making them a natural target for bioremediation of these PFAS chemicals, which are long carbon backbones surrounded by fluorine. While the 2020 USAFA iGEM team successfully identified the enzymes and provided rudimentary evidence they worked, their work left many gaps and was difficult to replicate. This team tackled that issue, by doing a more thorough analysis of the microbial genome to identify additional dehalogenases, purifying each individually and cloning them separately to provide a more controlled interrogation of their activity.

## Design

![](https://static.igem.org/mediawiki/2021/9/96/T--USAFA--DeHa_sequence_alignment.png)

Each iteration of the engineering process began with a purposeful, genomics-based design. Previously documented enzymes were blasted against the NCBI database and our newly sequenced isolate to identify five total dehalogenase enzymes in the  *Delftia acidovorans* genome. These enzymes were modeled to determine binding pockets and binding affinities for different fluorinated compounds, ensuring potential activity before starting the cloning process. Once our team confirmed the enzyme sequence, structure and ligand binding sites, we determined our cloning strategy.

## Build

![](https://static.igem.org/mediawiki/2021/9/94/T--USAFA--work_flow.png)

While these dehalogenase enzymes occur naturally in  *Delftia acidovorans*, this organism does not grow as quickly or robustly as other organisms such as  *Escherichia coli*. Additionally, we wanted to be able to evaluate our enzymes both individually and in tandem, thus they needed to be inserted into a new organism without native dehalogenases. Our team considered the plasmid backbone and dehalogenase insert to determine our strategy in terms of PCR amplification, restriction digests, ligation, transformation and confirmation sequencing. Once we successfully transformed the plasmid into  *E. coli*, we evaluated our construct to see if it lived up to the name: Defluorine Machine!

## Test

![](https://static.igem.org/mediawiki/2021/5/58/T--USAFA--fungi_results.jpeg)

A deliberate testing process proved instrumental in determining if the engineered parts were successful. From the beginning or our initial trials, it became apparent microorganisms tolerate and even proliferate in the presence of these potentially harmful PFAS chemicals. After their growth was confirmed, the question remained: did our enzymes remove fluorines from fluorinated chemicals, and if they did, how would we know? The team tried multiple approaches, but arrived at two basic techniques: a fluoride ion probe and LC-MS/MS. The team selected the fluoride ion probe for ease of use and timeliness of results, but the probe is only useful if fluoride is (1) being released during the process and (2) staying around long enough to be measured. The LC-MS/MS requires a higher learning curve and expensive, specialized equipment; however, it also tells us a more complete story of each reaction, allowing us to better characterize these complex enzymatic relationships.

## Learn

![](https://static.igem.org/mediawiki/2021/b/b3/T--USAFA--ELISA_Results.jpeg)

Each enzyme we identified and subsequent part built included its own oddities and lessons learned. Additionally, each technique implemented required its own set of controls and a deep understanding of the underlying assumptions for each experiment. Ultimately, the team used the first dehalogenase to troubleshoot protocols and build reliability and repeatability into the protocols, to pave the way for minor tweaks during subsequent iterations. Although we deftly navigated the engineering process this year, our work is far from done. We have enzymes capable of defluorination, but PFAS remains a large problem. Our next phase of development will bring us back to the design phase to answer the questions: how can we make this better? Faster? More efficient? Cheaper? At scale?

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# Team:USAFA/Experiments

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![](https://static.igem.org/mediawiki/2021/b/bb/T--USAFA--experimentsheader1.jpg)

## USAFA iGEM 2021 Experiments

This year, we performed a range of experiments to help further develop our project. This list includes basic procedures used most commonly in labs and developed procedures to test for defluorination. The pdf to the left contains all of our experiments and scrolls separately on the page. If you have further inquiries about any of our experiments, please do not hesitate to reach out at igem.usafa@gmail.com, or by clicking the link below.  ![](https://static.igem.org/mediawiki/2019/thumb/f/fe/T--NCKU_Tainan--Mail_icon.png/600px-T--NCKU_Tainan--Mail_icon.png)

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# Team:USAFA/Results

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](https://static.igem.org/mediawiki/2021/6/68/T--USAFA--resultsvidMP4V2.mp4)

## 1. Identify Dehalogenases

Our engineering success started with successful identification of five novel dehalogenase enzymes (DeHa 1-5) in the newly isolated and sequenced strain  *Delftia acidovorans* D4B. The 2020 USAFA iGEM Team had already identified DeHa 1 and DeHa 2 from a BLAST search of  *Delftia acidovorans,*  but our 2021 team revealed  *D. acidovorans* strain D4B contains the novel enzymes DeHa 3, 4, and 5.  ![](https://static.igem.org/mediawiki/2021/8/89/T--USAFA--D4B_sequence.png)

## 2. Model Putative Dehalogenases with PFAS Ligand

The protein sequences for DeHa 1-5 were then modeled using supercomputer molecular dynamics simulation software to determine the structure of the enzymes. These models were also utilized to identify potential PFAS-binding pockets.  ![](https://static.igem.org/mediawiki/2021/f/fa/T--USAFA--DeHa_together.png)

## 3. Clone and Purify Dehalogenases

Once we confirmed the sequences and predicted structures of DeHa 1-5, we proceeded with the expression, purification, and activity testing of these enzymes. We cloned DeHa 1-5 into individual pET15b expression vectors, and then our enzymes were expressed in  *Escherichia coli.* We then purified DeHa 1-5.  ![](https://static.igem.org/mediawiki/2021/3/36/T--USAFA--Purified_DeHa.jpg)

## 4. Confirm Activity

The final step was confirming the enzymatic activity of our dehalogenases. We used a calibrated fluoride probe and fluorine NMR to detect PFOA degradation. Fluorine NMR revealed both DeHa 1 and DeHa 5 are capable of cleaving off the terminal trifluoromethyl group of PFOA. These experiments produced the first evidence we may have created the DeFlourine Machine!

![](https://static.igem.org/mediawiki/2021/a/a4/T--USAFA--DeHa_trifluoromethyl.png)

# Supplemental Results

While initial data supports PFAS degradation by DeHa 1 and DeHa 5, data gathered with the fluoride probe and fluorine NMR revealed DeHa 2 and DeHa 4 are also enzymatically active! DeHa 2 and DeHa 4 defluorinate the monofluorinated compound ethyl fluoroacetate, as shown in the fluorine NMR spectra below.

![](https://static.igem.org/mediawiki/2021/3/3a/T--USAFA--Fluoride_NMR.jpg)

Initial kinetic studies of DeHa 2 and DeHa were then performed to show the monodefluorination kinematics of these enzymes, as seen below.

![](https://static.igem.org/mediawiki/2021/2/2e/T--USAFA--DeHa2_EFAC.jpg)

Kinetic study of DeHa 4 defluorinating ethyl trifluoroacetate:

![](https://static.igem.org/mediawiki/2021/6/65/T--USAFA--DeHa4_EFAC.jpg)