Part:BBa_K5114227
Human liver fatty binding protein-GFP conjugation coding sequence
The FAB_GFP conjugation sequence was originally characterized by Mann and Berger in 2013 and used to test for absorbance upon titration of PFOA. FAB is a human liver fatty acid binding protein found within the cytoplasm of human liver cells and mediates the intercellular transport of various fatty acids.
Usage and Biology
The FAB_GFP conjugation is oriented in a way that creates a beta-barrel in the folding of the GFP. This beta barrel allows for water inflow, causing the chromatophore in GFP to be disrupted and therefore halting fluorescence. However, when FAB binds to its ligand (natively to fatty acid-like molecules and, in our case, PFOA), the confirmation will change, greatly reducing the inflow of water and increasing fluorescence to a theoretically detectable level.
Below are images of the FAB_GFP conjugated protein and its structure:
We, Team GCM-KY 2024, decided to use a FAB_GFP complex for PFAS detection for a multitude of reasons. Primarily, PFAS has a structure quite similar to a fatty acid, thus warranting the possibility that PFAS may bind with a FAB molecule. We ran a reverse screening search on PFOA’s smile string to confirm this, resulting in high binding probabilities with FAB. We also conducted an exhaustive docking study via Autodock Vina and Amber. The docking study revealed a high likelihood that PFOA can bind in the FAB domain; to test how strong the bind was, the best pose predicted by Autodock Vina was used and a simulation under explicit water solvent conditions was used. After the simulations were completed, a Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) method was used to calculate the Gibbs free energy of binding. The result was -13 kcal/mol, meaning 13 kcal of energy would be required to dissociate PFOA from FAB. Which indicates strong, drug-like binding to the FAB domain. This study validated our hypothesis that Fatty Acid Binding Proteins can be used as a PFAS detector.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
In order to test the FAB_GFP protein, we put the sequence under the influence of a constitutive promoter inside of a plasmid with Kanamycin resistance. Then, we transformed this plasmid into competent DH5-alpha Escherichia coli, performing both gel electrophoresis and blue-white screening to ensure proper transformation. We then took successful colonies, grew them, and exposed them to different PFAS concentrations.
When analyzing our results through a fluorometer, we saw little change in the fluorescence of the solution, suggesting that there was either inadequate amounts of PFAS, unsuccessful transformation, or that our theory of PFAS binding to the FAB_GFP complex was faulty altogether. More experimentation is needed to confirm the root of the cause.
Possible Uses for Other Teams
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). 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.
References
Mann, M. M., & Berger, B. W. (2023, September 13). A genetically-encoded biosensor for direct detection of perfluorooctanoic acid. Nature News. https://www.nature.com/articles/s41598-023-41953-1
Smathers, R. L., & Petersen, D. R. (2011, March 1). The human fatty acid-binding protein family: Evolutionary divergences and functions - human genomics. BioMed Central. https://humgenomics.biomedcentral.com/articles/10.1186/1479-7364-5-3-170
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