Part:BBa_K5114228
Expression device for BBa_K5114227 (hlFAB-GFP)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite sequence was created by GCM-KY 2024 to test the potential of the FAB_GFP Conjugated protein (https://parts.igem.org/Part:BBa_K5114227) in detecting PFAS. This composite part contains the sequence p_Const-RBS (Ribosomal Binding Site)-FAB_GFP-Double Terminator.
The FAB_GFP conjugation sequence was originally characterized by Mann and Berger in 2023 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.
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.
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 supported our hypothesis that Fatty Acid Binding Proteins can be used as a PFAS detector.
Characterization
Labwork
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.
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. These mutations are potential points of future experimentation.
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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