iGEM 2021 — Team CTR_Alberta_Canada

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# Team:CTR Alberta Canada/Notebook

# For Future Consideration...

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# Team:CTR Alberta Canada/Engineering

# A) ENGINEERING DESIGN CYCLE - SUCCESSES

The engineering design cycle was demonstrated throughout the parts, device and systems-level of our project. This process enabled our team to address solutions and improve from our failures with the goal of developing a project that can have meaningful impacts.

![Image of engineering design cycle](https://static.igem.org/mediawiki/2021/f/f4/T--CTR_Alberta_Canada--Engineering_cycle_CTR.png)

### Design

Oxybenzone is a chemical compound found in sunscreens that serves as an active ingredient to block ultraviolet (UV) rays. Concerns around the rising impacts of oxybenzone on coral bleaching and the human endocrine system led to the designing of this project - detection of oxybenzone concentrations through the use of a two plasmid system engineered for *S. cerevisiae*. The development of an oxybenzone detection mechanism will enable us to fully understand the extent of the problem and determine what further steps are needed to attempt to solve this issue.

The system we are using to implement the testing of oxybenzone concentration levels in water is based upon the Field-Ready Electrochemical Detector (FRED), which was developed and used by mentors at FREDsense technologies. Collaboration and mentorship with FREDsense technologies were crucial to designing our project and guiding the direction of our research. Extensive research has been done on Hawaii's sunscreen bans, different estrogen disruptors, different sunscreen alternatives, differences between organic and inorganic sunscreen, etc. to ensure our project is designed with accuracy and a basis of knowledge.

Certain members of our team also conducted several interviews with different specialists, helping to further our project by giving our team a perspective of our problem through a specialist lens.

Originally our project utilized the part BBa\_K1732015, which would be able to detect oxybenzone concentration via an estrogen receptor ligand-binding domain attached to a T7 RNA polymerase, inducing a T7 promoter on a second plasmid. Unfortunately, we couldn’t find anything on the author or any further data on this part, so decided to pivot and use a different system for our oxybenzone detection project.

### Build

Using a synthetic fusion protein estradiol detection system developed for *S. cerevisiae* by Aranda-Diaz et al. (2017) as a reference, a two plasmid system was designed to detect oxybenzone through its binding to a human estrogen receptor (hER). When oxybenzone is present, it will bind to a protein expressed by the first plasmid (Plasmid One) that induces the promoter of the second plasmid in the system (Plasmid Two), leading to the expression of the  [LacZ gene](https://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00073) .This gene codes for the synthesis of the enzyme beta-galactosidase, which breaks the substrate para-aminophenol-beta-d-galactopyranoside (PAPG), down into galactose and para-aminophenol (PAP), producing an electrochemical signal which can be measured and analysed to determine oxybenzone concentration (Figure Below).

![](https://static.igem.org/mediawiki/2021/c/cc/T--CTR_Alberta_Canada--Figurie1_ENGINEERING.png)

The system for implementation includes a cartridge containing the engineered *S. cerevisiae* and a para-aminophenol-beta-d-galactopyranoside (PAPG) substrate. Water is collected and added to the cartridge, which is then placed in a FRED device. From there, the device incubates the test and the results will be available for analysis.

### Our Parts

* [BBa\_K3856000](http://parts.igem.org/Part%3ABBa_K3856000)
* [BBa\_K3856002](http://parts.igem.org/Part%3ABBa_K3856002)
* [BBa\_K3856003](http://parts.igem.org/Part%3ABBa_K3856003)
* [BBa\_K3856004](http://parts.igem.org/Part%3ABBa_K3856004)

[Parts Page](https://2021.igem.org/Team%3ACTR_Alberta_Canada/Parts)

### Test

The DNA for our two plasmids as well as materials needed to test our project are ready to go, but unfortunately due to COVID-19 restrictions, we have been unable to go into a lab to further prove the accuracy of our project. As soon as we are able, additional lab work to further prove the effectiveness of the developed plasmids will need to be done to expand on and develop this project. For testing purposes, samples of engineered *S. cerevisiae* will be introduced to water samples with specific, known quantities of oxybenzone concentrations.

To verify the theory of our project, 3D modelling of the GEM protein, the hER and oxybenzone have been done using the pyMOL modelling software. The aligning and binding of oxybenzone to the hER, as well as our GEM protein (containing the hER) has been done to confirm the theory behind our system.

### Learn

Through the design, build and test stages of our engineering process we learned many things, including the fact that the hER is unable to be expressed in *E. coli*. Due to this, our two plasmid system was engineered for expression by *S. cerevisiae* as the detection system relies on a human estrogen receptor (hER) to detect oxybenzone concentrations. Our project was designed to use *E. coli* for replication of our plasmids, where they will be transformed into *S. cerevisiae* for use in our detection system. As further testing and modelling occur, we will continue to learn more about our solution and find ways to make our project more accurate and specific in solving the problem of oxybenzone contamination.

### Design

Further work on the design of our system implementation will occur once testing and modelling have proven our project to be effective.

# B) DESIGN THINKING INNOVATION CYCLE - SUCCESSES

We used design thinking to successfully cycle through our human practices. Please visit our Human Practices for a full description.

[Human Practices](https://2021.igem.org/Team%3ACTR_Alberta_Canada/Human_Practices)

# C) MODELING DESIGN CYCLE - SUCCESSES

We iterated through modeling cycles towards 3D design successes. Please visit our Model page for a full description.

[Model](https://2021.igem.org/Team%3ACTR_Alberta_Canada/Model)

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# Team:CTR Alberta Canada/Experiments

# For Future Consideration...

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# Team:CTR Alberta Canada/Results

# For Future Consideration...