iGEM 2024 — Team iea

## Notebook Folder

Notebook Links

The Notebook Folder contains a pdf of the Experiment Group's Benchling notes, protocols, and pictures.

<https://tools.igem.org/uploads/teams/5035/lab-notebook>

<https://static.igem.wiki/teams/5035/lab-notebook/iea-experiment-group-notebook-2024-06-02-2024-09-18-etr-uw9cwzeb.pdf>

<https://static.igem.wiki/teams/5035/lab-notebook/experiment-group-notes-1.pdf>

<https://static.igem.wiki/teams/5035/lab-notebook/iea-protocols.pdf>

Inluded are the following protocols used in the enzyme design, and the expression classifier projects:

PCR Method

Purification(SPRI) Method

PURE (TXTL) Method

TXTL Method

Electrophoresis Method

TXTL Purification Method

Mini-Protean TGX stain-free Precast Gel Method

TXTL Under Denaturation Method

TXTL + Cofactors Method

Cofactor Reconstitution (Nakamura) Method

PCR Optimization Method

KAPA HiFi PCR Method

RepliQa PCR Method

Phusion PCR Method

GxL PCR Method

E-Gel EX 4% Protocol

Bioanalysis Machine Protocol

### Summary of Activities January 2024 - July 2024

For the first months, we spent a lot of time (about 6-10 hours per week) on computation, learning and trying various techniques, refiniing computing skills, adapting, creating, and modifying software to design enzymes. We also learned new wetlab skills and protocols. When the summer started we spend a lot of time (10-12 hours per day) on computation (to create novel enzymes) and working on the expression classifier, because we discovered that novel enzymes are very hard to express in any circumstances.

Experiment Group Notebook

![Troy and Lucas Computing](https://static.igem.wiki/teams/5035/team-pics/lucas-and-troy-computing.webp)

Troy and Lucas Computing

### Summary of Activities July 2024 - September 2024

In July, we had acquired most of the skills we needed for our wetlab work, and spent most of the time in the lab, in addition to computation work (10-12 hours per day, 5 days per week). All the wetlab work is documented in the attached Lab Notebook, and briefly summarized below.

![Ray and Jael at bench in Thomson Lab](https://static.igem.wiki/teams/5035/team-pics/ray-and-jael-working-at-igem-bench.webp)

Ray and Jael at bench in Thomson Lab

### Week by Week Brief Summaries

**Intro:** The main focus of the wet lab was to bring the dry lab’s vision and progress to life.

### Weeks 1 - 2: July 17 - July 24

Throughout the first two weeks of the experimentation, progress focused heavily on the preliminary expression of candidate enzymes. This included DNA amplification, purification, and cell-free mixture expression. Additionally, results were measured through gel electrophoresis and Qbit analysis.

### Weeks 3 - 4: July 24 - August 7

The third and fourth weeks of experimentation focused on the improvement of said expression as the preliminary results were inadequate. The basis of these weeks was expressing T7RdhA to see what was needed for the proper expression of main candidate enzymes.

### Weeks 5 - 6: August 7 - August 28

The fifth and sixth weeks of experimentation were arguably some of the most tedious and strenuous of the project process. In these weeks, long processes of PCR and purification were completed for new enzymes in addition to T7RdhA.

### Weeks 7 - 9: August 28 - September 13

The longest stretch of repeated and complicated processes came from the seventh through eighth weeks. During this time, the processes for enzyme expression became streamlined and thorough enough for results to be effective and efficient. The completion of PCR optimization, cofactor reconstitution, and electrophoresis analysis proved to be effective methods of expression.

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# Engineering Success

Through iterative cycles of computational design, testing, and optimization, the team successfully created the A6T7 Chimera PFAS-degrading enzyme and improved protein expression techniques despite initial challenges. The first design success we detail in the main page of our part, https://parts.igem.org/Part:BBa\_K5035003. We will use the remainder of the page to describe an early challenge we overcame with protein expression.

![Home Image](https://static.igem.wiki/teams/5035/unnamed-2.gif)

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One of the design challenges we overcame early on in our iGEM journey was expressing one of the dehalogenases we investigated, T7RdhA. Expressing it in a cell-free extract led to formation of an insoluble pellet (shown below). Solubilization of the pellet with guanidium hydrochloride followed by running an SDS-PAGE gel revealed the pellet to be misfolded, aggregated protein (gel image).![insoluble insoluble-t7rdha](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/insoluble-t7rdha.webp)

Pellet of insoluble aggregate of T7RdhA formed at the bottom of the tube

![T7RdhA Misfolding](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/t7rdha-was-misfolding.webp)

Denatured T7RdhA was successfully purified from a cell lysate solution, as shown in this protein electrophoresis gel.

## Addressing T7RdhA Misfolding: Testing Cofactor Infusion and Nakamura Method for Protein Folding, Leading to Successful Expression

To fix this issue, the team devised two potential strategies: infusing the expression solution with the cofactors so that as the protein is translated, the cofactors integrate into the structure of the protein or express T7RdhA without cofactors and then denature the T7RdhA inclusion bodies and renature them with cofactors present to reconstitute them into their structure. We decided to attempt both strategies simultaneously. The first strategy did result in the absence of the inclusion body pellet; however, when purified, only a faint band or no band at all at the correct size was observed, leading us to conclude that this method only resulted in minimal yield. The second method, we dubbed the Nakamura method after the primary author of the paper that details it, resulted in a much clearer band at the correct size, the downside being the complexity and lengthiness of the protocol.

![Nakamura Gel](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/nakamura-gel.webp)

The Nakamura protocol results in a higher yield of refolded enzymes than previous folding methods.

## Successful Expression of O68252, A6T7 Chimera, A0A7C3HHU1, A0A2E5NSL8, and A0A419DFF0 for Off-Site Substrate Evaluation

With this success, we went on to express O68252, A6T7 Chimera, A0A7C3HHU1, A0A2E5NSL8, and A0A419DFF0 and send these purified enzyme solutions off-site to be tested against a PFAS substrate.

![gel1](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/protein-gel-t7rdha.webp)
![gel1](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/protein-gel-selectedvariants-igem-2-9-19-24-1.webp)
![gel1](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/protein-gel-a6t7-chimera-2024-09-22-at-11-16-09-pm-1.webp)

The A6T7 Chimera, O68252, A0A7C3HHU1, A0A2E5NSL8, and A0A419DFF0 were successfully expressed in MyTXTL and reconstituted with the necessary iron, sulfur, and BVQ cofactors.

## PFAS-Degrading Parts Entered into Parts Registry

See our [Results](https://2024.igem.wiki/iea/results), [Contribution](https://2024.igem.wiki/iea/contribution), [Notebook](https://2024.igem.wiki/iea/notebook), and [Project Description](https://2024.igem.wiki/iea/description) pages for details of the iterative process, and links to our parts.

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# Our Experiment

See our [Description](https://2024.igem.wiki/iea/description), [Notebook](https://2024.igem.wiki/iea/notebook), [Parts](https://2024.igem.wiki/iea/parts) pages for more information.

## Overview

The team combines AI-driven protein design with experimental validation, assessing the expressibility of the computationally generated enzymes in a cell-free system to ensure feasibility for lab testing. Successfully expressed enzymes are then sent to a specialized lab to confirm PFAS degradation activity, and as input for an expression classifier. From that expression classifier, enzymes predicted to be expressible in TXTL are then sent to the wet lab team to be expressed in TXTL. Whether the enzyme is successfully expressed or not is experimentally identified to ensure enzyme yield and strengthen future expression predictions. Then lastly, we will send our enzymes to a separate lab to test them on a PFAS substrate. Successfully expressed enzymes will be sent to a professional lab that can handle PFAS. The lab’s team of experts will use our enzymes in a PFAS degradation assay to validate their catalytic activity. If degradation is not observed, we will alter our generation procedure and our prediction mechanisms.

## Discovery of A6RdhA

The team discovered the corrinoid iron-sulfur reductive dehalogenase A6RdhA. A6RdhA was identified within *Acidimicrobium sp. Strain A6*, a soil-dwelling microbe that, when incubated with a PFOA substrate, could partially defluorinate PFOA's structure. PFOA is a legacy PFAS species that was recently regulated by the EPA in 2024 down to 4 parts per trillion (ppt), the minimal detectable limit.

## Addressing T7RdhA Misfolding

To fix this issue, the team devised two potential strategies: infusing the expression solution with the cofactors so that as the protein is translated, the cofactors integrate into the structure of the protein or express T7RdhA without cofactors and then denature the T7RdhA inclusion bodies and renature them with cofactors present to reconstitute them into their structure. We decided to attempt both strategies simultaneously. The first strategy did result in the absence of the inclusion body pellet; however, when purified, only a faint band or no band at all at the correct size was observed, leading us to conclude that this method only resulted in minimal yield. The second method, we dubbed the Nakamura method after the primary author of the paper that details it, resulted in a much clearer band at the correct size, the downside being the complexity and lengthiness of the protocol.

[![Nakamura Gel Image](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/nakamura-gel.webp)](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/nakamura-gel.webp)

View Nakamura Gel Image

## A6T7 Chimera

The A6T7 Chimera reconstructed the A6RdhA fragment and was successfully expressed in a cell-free extract.

* **Fig. 4|a**: The A6T7 chimera was produced by reconstructing the missing C-terminus of the original A6RdhA fragment with the C-terminus of the T7RdhA enzyme. The addition of this C-Terminus reformed the active site of the A6RdhA fragment making it more likely to be capable of binding essential cofactors.
* **Fig. 4|b**: The A6T7 Chimera was successfully expressed in MyTXTL and reconstituted with the necessary iron, sulfur, and BVQ cofactors.

## Successful Expression and Off-Site Evaluation

With this success, we went on to express O68252, A6T7 Chimera, A0A7C3HHU1, A0A2E5NSL8, and A0A419DFF0 and send these purified enzyme solutions off-site to be tested against a PFAS substrate.

## Selection of Promising Novel Enzymes

Of the sequences produced, 3 of the most promising generated enzymes in terms of structure and novelty were chosen to be expressed in MyTXTL. The DNA coding for these novel enzymes was ordered and will be prepared for expression.

## Affinity Scores of Candidate Enzymes

Here are the affinity scores of candidate enzymes as they relate to PFAS degradation:

[![Affinity Scores ](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/affinity-scores-of-candidate-enzymes.webp)](https://static.igem.wiki/teams/5035/flowcharts-gels-graphs/affinity-scores-of-candidate-enzymes.webp)

Affinity scores

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# Results

The A6T7 Chimera showed high predicted affinity for PFAS ligands, and six candidate enzymes were successfully expressed and sent off-site for further testing on PFAS substrates. We made an expression classifier (or regressor).

![Home Image](https://static.igem.wiki/teams/5035/unnamed-2.gif)

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## Project Achievements

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### Parts

* [https://parts.igem.org/Part:BBa\_K5035003](https://parts.igem.org/Part%3ABBa_K5035003)
* [https://parts.igem.org/Part:BBa\_K5035002](https://parts.igem.org/Part%3ABBa_K5035002)
* [https://parts.igem.org/Part:BBa\_K5035004](https://parts.igem.org/Part%3ABBa_K5035004)
* [https://parts.igem.org/Part:BBa\_K5035005](https://parts.igem.org/Part%3ABBa_K5035005)
* [https://parts.igem.org/Part:BBa\_K5035006](https://parts.igem.org/Part%3ABBa_K5035006)
* [https://parts.igem.org/Part:BBa\_K5035007](https://parts.igem.org/Part%3ABBa_K5035007)
* [https://parts.igem.org/Part:BBa\_K5035008](https://parts.igem.org/Part%3ABBa_K5035008)
* [https://parts.igem.org/Part:BBa\_K5035009](https://parts.igem.org/Part%3ABBa_K5035009)
* [https://parts.igem.org/Part:BBa\_K5035010](https://parts.igem.org/Part%3ABBa_K5035010)

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### Enzyme Design Software

We harnessed protein large language models (pLLM) to generate novel PFAS degrading enzymes, and then computationally validated our enzyme’s activity with PFAS. Through many computational design, testing, and optimization runs, the team successfully created the A6T7 Chimera PFAS-degrading enzyme and 3 novel AI-designed dehalogenases. See [Part:BBa\_K5035003](https://parts.igem.org/Part%3ABBa_K5035003) and our [Model](https://2024.igem.wiki/iea/model) page for more information.

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### Expression Classifier

We designed an expression classifier applicable to projects beyond our own, enhancing protein expression prediction for our, as well as future research. See our [Software](https://2024.igem.wiki/iea/software) page for more information.

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### Experimental Results

We were able to express our generated enzymes in the wet lab, later sending those enzymes to a separate lab to test them on a PFAS substrate. We are still waiting on results from our collaborator, but hope to be able to provide updates at the Jamboree. The success of our expression experiments allows us to make more informed computational predictions so that we can tailor our process to optimally create novel PFAS-degrading enzymes in the future.

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### Integrated Human Practices

By identifying different stakeholder groups including PFAS scientific experts, participants in the PFAS remediation industry, regulators and water treatment facilities, and community stakeholders, we could locate and engage with many experts. These experts influenced the direction of our project, resulting in pivots, enhancements, and validations at various stages.

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