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GCM-KY 2024 usage

Transformation and Selection

We printed the expression device (BBa_K5114228) in the pUC57-Kan plasmid from Genscript.

We performed a restriction digest with SapI and ran the results on a gel. The bands that were produced were as expected. Construct 2 is the hlFAB-GFP expression device. The ladder is 1 kb per band.

These were transformed into DH5-alpha E. coli and cultured on Kanamycin plates containing X-gal. The presence of colonies and positive blue/white screening results indicate successful transformation.

Fluorescence Testing

After confirming its presence, the transformed bacteria were exposed to different PFOA concentrations. Fluorescence levels were taken at regular time intervals. The data is contained in the image below:

As seen by the heat map above (in the bottom two rows), increasing concentration exposure did have a slight impact on the fluorescence. As the concentration increased, there was a slightly increased amount of fluorescence intensity. However, when compared to the fluorescence intensities of the LB Broth, which was intended to act as a negative control, there is an indication that there may be errors in the measurement of the fluorescence itself. In order to investigate this more, a proper graph was created.

Overall, based on lab results, we did not observe significant differences for any of our hlFAB-GFP replicates as we increased the amount of PFOA we exposed them to. When we consulted the original authors, they suggested that we use an inducible T7 promoter to ensure enough protein is produced. Additionally, when the sequence of the protein we reconstructed from the original publication was compared to the sequence used by the authors, we identified several substitutions and deviations from their sequence. We did not have time to make improvements in the lab, so this is left as a direction of future work.

Molecular Dynamics Simulations

After testing hlFAB in the lab to detect PFAS, we wanted to explore whether modifications could optimize its performance and then test the new hlFAB variants in the lab. To do this, we used rotamers, a feature in ChimeraX, to swap specific residues—for example, replacing residue 351 (Threonine) with Serine. You can explore the specifics of our mutations on the results page of our website. The mutated structures were then processed through our MMPBSA pipeline, also available in our repository, to calculate the dissociation constant (Kd). If the Kd value was higher than -13 kcal/mol, the mutation was considered beneficial and could potentially enhance the lower detection limit of hlFAB.

Applications of BBa_K5114227

Although we didn’t see much success in the results of this protein within E. coli, it’s possible that other teams could use this part for either fatty acid binding or for binding with PFOA (and other types of PFAS).

Future pathways/ways it could be optimized or improved

More testing of the corrected sequence is needed to properly determine the real functionality of this protein. We will also need to determine if a stronger, inducible promoter would work better than the constitutive promoter (Pconst-BBa_J23100) that we used. Molecular dynamics also identified possible point mutations in the PFOA binding site that could be used to improve the binding affinity.

User Reviews

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