iGEM 2021 — Team Stanford

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stanford/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stanford/Engineering

[![](https://static.igem.org/mediawiki/2021/1/11/T--Stanford--citrusafelogo.png)](https://2021.igem.org/Team%3AStanford)

# 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

![](https://static.igem.org/mediawiki/2021/5/5f/T--Stanford--engineering-success.png)

#### 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.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stanford/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Stanford/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)