Our Experiment

See our Description, Notebook, 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.

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