Team:Stanford/Engineering


Engineering Success

What was our engineering process in designing Citrus Safe?

Design Iterations Include:

  • Engineering a modified CYP3A4 enzyme into the gut microbiome to serve as a competitive inhibitor against drug molecules
  • Using alphafold to make candidate substitutes for the alternative CYP3A4 with an active binding site incapable of metabolizing the drug molecules
  • Isolating citrus fungus (citrus paradisi) from grapefruit trees in an attempt to metabolize furanocoumarin-class molecules
  • Taking a pre-existing CYP enzyme (CYP6B1) from black swallowtail butterflies known to digest furanocoumarin molecules and synthesizing the same expression pathway in baker’s yeast, then e. Coli
  • Designed plasmid to insert in yeast, could not get quantification experiments done



    • Cycle 1

    The engineering of our enzymatic expression system began by parsing through prior literature to learn more about the human CYP3A4 enzyme’s function, ubiquity, and ability to bind to different classes of drugs. Several modifications to the enzyme were discussed; we could simply produce more of the enzyme to increase bioavailability, an allosteric inhibitor that competes with the furanocoumarin molecules, and finally settled on an alternative CYP3A4 enzyme that could form the lock-and-key mechanism with both furanocoumarin and drug molecules, but would be functionally inert with furanocoumarins. Using the PyRosetta script with PyMOL visualization, we modified multiple components to find combinations of conformational macrostates and their oriented binding sites. With Alpha Fold, we attempted to produce predictive models with modified binding sites that would be better adapted for drug molecules than furanocoumarin molecules. We collaborated with the UC Davis iGEM 2021 team in which we produced a stochastic simulation of a furanocoumarin ligand docked into the CYP3A4 active site as well.


    Cycle 2

    Instead of merely physically reducing the inhibitory effect of furanocoumarin molecules, we decided on an approach to search for a biological component that could degrade the furanocoumarins. We looked into ecological food chains to find natural predators of allelochemical-containing grapefruit fruit bodies, more specifically the fungus citrus x paradisi. This led to the design of an autoregulatory circuit that degrades furanocoumarins with a specific combination of CYP-class enzymes specifically catered to breaking down the primary furanocoumarin molecules found in grapefruit. We established a protocol and experiment to quantify furanocoumarin levels and set these values to be our positive control to account for natural degradation. Going further down this cycle, we quickly realized that to isolate the responsible pathways behind citri x paradisi’s ability to cohabitate grapefruit bodies would be a biological experiment, not necessarily a genetic engineering approach.


    Cycle 3

    We discovered a eukaryotic gene in black swallowtail butterflies that encodes for CYP6B1 enzyme, which is homologous to CYP3A4 but with the distinct phenotype of being able to metabolize furanocoumarins. Through Benchling, we constructed e. Coli plasmids with the CYP3A4 gene of interest and codon optimized for expression in Saccharomyces cerevisiae, a high-turnover eukaryotic gut microbe that colonizes the gut in relatively small quantities. In this iterative third cycle, we discovered in literature that gene expression for CYP6B1 was significantly heightened coupled with a reductase enzyme sourced from fruit fly Drosophila melanogaster. Going forward, we discovered an even better candidate host organism that propagates in the human digestive tract at much higher percentage than Baker’s Yeast, and that eukaryotic CYP6B1 has been successfully expressed in e. Coli before. We produced an e. Coli plasmid that had a modified CYP6B1 gene sequence without the N-terminus, trans-membrane tail unique to the eukaryotic homolog by isolating it through the TMHMM Server 2.0 and replacing it with a Bacillus tail sourced from literature. With the quantified furanocoumarin positive control levels, we aim to test differentially expressed protein levels of CYP6B1 and compare the levels to determine metabolic enzyme activity of CYP6B1 integrated in e. coli.


    Setbacks due to COVID-19

    While society seems to be on the path to a more “normal” way of life, the impacts of the coronavirus are still extremely prevalent and have caused many experimental setbacks throughout the past few months. At Stanford University, our team was forced to jump through many logistical hoops to even have access to buildings and labs on campus, cutting the time we had to work on our project by a significant amount. In addition to our reduction of in-lab time, COVID-19 caused many of the reagents and materials we required for our project to be delayed. Without the necessary reagents and materials, our experiment was further delayed, putting more stress on our overall timeline. While we were able to overcome many of the unforeseen obstacles that were thrown in front of us, it is important to recognize that setbacks unfortunately still occurred, and we did not get through each cycle that we had planned.