iGEM 2021 — Team RHS-Calgary

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# Team:RHS-Calgary/Notebook

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Notebook

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* [Home](https://2021.igem.org/Team%3ARHS-Calgary)
* Project

  + [Description](https://2021.igem.org/Team%3ARHS-Calgary/Description)
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  + [Contribution](https://2021.igem.org/Team%3ARHS-Calgary/Contribution)
* [Notebook](https://2021.igem.org/Team%3ARHS-Calgary/Notebook)
* Wet Lab

  + [Experiments](https://2021.igem.org/Team%3ARHS-Calgary/Experiments)
  + [Results](https://2021.igem.org/Team%3ARHS-Calgary/Results)
  + [Engineering Success](https://2021.igem.org/Team%3ARHS-Calgary/Engineering)
  + [Parts](https://2021.igem.org/Team%3ARHS-Calgary/Parts)
  + [Safety](https://2021.igem.org/Team%3ARHS-Calgary/Safety)
  + [Sustainability](https://2021.igem.org/Team%3ARHS-Calgary/Sustainable)
* Human Practices

  + [Human Practices](https://2021.igem.org/Team%3ARHS-Calgary/Human_Practices)
* Team

  + [Collaboration](https://2021.igem.org/Team%3ARHS-Calgary/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3ARHS-Calgary/Attributions)
  + [Members](https://2021.igem.org/Team%3ARHS-Calgary/Team)

* [Home](https://2021.igem.org/Team%3ARHS-Calgary)
* Project

  + [Description](https://2021.igem.org/Team%3ARHS-Calgary/Description)
  + [Implementation](https://2021.igem.org/Team%3ARHS-Calgary/Implementation)
  + [Contribution](https://2021.igem.org/Team%3ARHS-Calgary/Contribution)
* [Notebook](https://2021.igem.org/Team%3ARHS-Calgary/Notebook)
* Wet Lab

  + [Experiments](https://2021.igem.org/Team%3ARHS-Calgary/Experiments)
  + [Results](https://2021.igem.org/Team%3ARHS-Calgary/Results)
  + [Engineering Success](https://2021.igem.org/Team%3ARHS-Calgary/Engineering)
  + [Parts](https://2021.igem.org/Team%3ARHS-Calgary/Parts)
  + [Safety](https://2021.igem.org/Team%3ARHS-Calgary/Safety)
  + [Sustainability](https://2021.igem.org/Team%3ARHS-Calgary/Sustainable)
* Human Practices

  + [Human Practices](https://2021.igem.org/Team%3ARHS-Calgary/Human_Practices)
* Team

  + [Collaboration](https://2021.igem.org/Team%3ARHS-Calgary/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3ARHS-Calgary/Attributions)
  + [Members](https://2021.igem.org/Team%3ARHS-Calgary/Team)

# Notebook

# team progress journal

**The story of GlucoLitter began back in 2019...**

**Thursday August 29, 2019:**

**-Finalized our team members**

**-Took at a Synthetic Biology guide book**

**September 18th, 2019:**

**-Created a project proposal outline**

**-Created a plan about our construct**

**September 27th, 2019:**

**-First wet lab experiment!**

**-Created an initial draft of our wiki**

**October 4th, 2019:**

**-Official Team organization**

**Human Practices:**

**-Amelia, Lane, Julia**

**Admin:**

**-Yali, Aashish**

**Business:**

**-Tony**

**-Aadesh**

**-Dayne**

**Wet Lab:**

**-YJ**

**-Emma D**

**-Rozina**

**-Noah A**

**Dry Lab:**

**-Waseem**

**-Tristalyn**

**-Arjun**

**-Noah J**

**Indeed, our project began in the early september of 2019. Since then, our team has been completely altered. All of the previous members have now graduated, and the torch of RHS-Calgary has been passed to a group of 8 new highschoolers.**

**In fact… back in 2019, our project was going to be research on the application of enzymes in eliminating nicotine addiction.**

**Fast forward to February 4, 2020…**

**We went back to the drawing board.**

**Some of our new ideas included an allergy detection system, crude oil removal, targeting contamination and the smells of protein shaker bottles, Effluent waste contamination in drinking water, and finally… feline diabetes.**

**February 12, 2020 - we began looking at the pros and cons of each project idea and what our team could contribute to the iGEM community**

**February to March, 2020 - investigating the iGEM requirements, determining funding goals, and brainstorming fundraising ideas**

**March 13th, 2020…**

**We started discussing other products that were currently available in the market and why ours was unique**

**The COVID-19 pandemic begins to shake the world. Our field trip to visit the university of lethbridge is cancelled.**

**During the time where we didn’t have access to the lab, we choose to focus on creating our wiki and establishing plans to do so**

**Fast forward to September, 2020…**

**Meet our New Member, Sophia Vig-Benko.**

**Unfortunately, many of our logs were lost in the transition between old members and new. We slowly began to lose the old members of our team and then joined our new members: Aarushi Bhargava, Tanisha Mattapalli, Kaylin Kritzler, Madison Kritzler, Diya Sharma, and Maya Jina.**

**We did, however, manage to have access to some of the interviews our old team members had done**

**Feb 3, 2021**

**Our Dry lab team began to work on an outline for the wiki and got feedback from all the other groups as to what information we would need for the wiki**

**Feb 5, 2021**

**An exciting time in any project… choosing a name**

**Here are our options:**

**Pissy Cat**

**GLOAT**

**Sprinkle and Tinkle**

**Glucolitter**

**Feline DIs**

**Sweet Pee**

**Pawtect**

**February 10, 2021**

**Weekly Meeting!**

**Created our very first iteration of the logo**

**February 26, 2021**

**HP begins working on storyboarding and scripting a promotional video for crowdfunding**

**Had a meeting with Sarah and Mr. Emond for feedback/mentoring**

**March 12, 2021**

**Tech Futures Challenge tomorrow!**

**We discussed the answers to some of the questions that we were expecting the judges to ask**

**Prepared for a question and answer session**

**Finalized Color scheme and logo**

**Mar 13, 2021**

**Exciting news… “WE GOT RUNNER UP FOR OUR CATEGORY!! This means that we won $1500! Great job to everyone who attended the pitch, worked on the promotional video, and participated in the Q & A session! We should all be super proud of ourselves and use this accomplishment as motivation to further develop our project and continue with the hard work!”**

**Mar 15, 2021**

**Interview with Mr. John Whitbread who provided us with some essential information for our project**

**Mar 31, 2021**

**Dry Lab decided to use nicepage in order to create the wiki**

**Got some advice from computer science teacher at our school**

**Apr 17, 2021**

**Mindfuel Lectures!**

**We attended many of these and collected notes from these presentations**

**May 6, 2021**

**Meeting with Dr. Laura Keffer Wilkes!**

**May 12, 2021**

**Custom Presentation template created by Aarushi Bhargava for consistent branding**

**Had a feedback session/meeting with the team**

**May 2021**

**Preparation for Tech Futures Challenge**

**Created our script and delegated different sections to each of our team members to research**

**Q/A Documents**

**May 26, 2021**

**Tech futures video filming**

**Did some research on gox**

**May 29, 2021**

**“As most of you know, we competed in the second part of the MindFuel Tech Futures Challenge (Project Prototypes) today! The awards ceremony just happened, and I'm pleased to share some good news with the team. We won overall runner-up which includes a $2500 prize. Additionally, we won top start-up project - this means we've been invited to go to a 5-day entrepreneurial summer camp at the University of Calgary this upcoming summer! This will be a great opportunity for our team, and will also be something we can discuss during the iGEM competition in the fall. Great job to everyone who worked on the video these past couple of days. I'm proud of our team, and let's keep up the great work!”**

**Jun 18, 2021**

**Interview with Emily Chorwood:**

**“I contacted Ms. Emily Chorwood and asked her what level of glucose in cat urine her clinic finds concerning - a very important part of our project. When blood glucose exceeds 10 mmol/L it is excreted in the urine. Anything above this amount is concerning. Normal blood glucose ranges between 3.3-8.3 mmol/L depending on the source you use. For the ranking system her clinic uses the information below:**

**+/- : 2.8 mmol/L (trace amounts, not always concerning)**

**1+ : 5.5 mmol/L**

**2+ : 14 mmol/L**

**3+ : 28 mmol/L**

**4+ : >55 mmol/L**

**With the test strips that her clinic uses, the urine will change the colour of the strip and that will show what grade/amount of glucose is in the urine. Her clinic doesn’t measure exact values. For those that are hyperglycemic and have glycosuria, her clinic sometimes runs additional blood work such as a fructosamine to get the precise value of glucose in the blood and to help monitor diabetic patients, typically cats over dogs. I have also contacted Dr. Nicole Howard, but I have not received a response yet. Hope this helps!”**

**Jun 23, 2021**

**Working on the preliminary safety form**

**Researched what temperature conditions our enzyme could survive in**

**Jul 15, 2021**

**Project Promo Storyboarding meeting**

**Script: Aarushi, Kaylin, Madison, Sophia**

**Filming and direction: Aarushi**

**Acting: everyone**

**Jul 16, 2021**

**Color change experiment**

**Jul 18, 2021**

**Project Promo Video Filming**

**Jul 22, 2021**

**Final Promo Video Draft Completed**

**Consent forms**

**Aug 25, 2021**

**Created a fundraising plan to gather money for the jamboree registration.**

**Got permission from our schools founder/principal**

**September 23, 2021**

**Created our iGEM team’s instagram account: renert.igem**

**Sep 25, 2021**

**Symposium with Dr. Laura Keffer-Wilkes and Lethbridge team**

**Oct 1, 2021**

**Finalized Safety form**

**Oct 3, 2021**

**cGEM competition - nationals!**

**Silver medal!**

**Creating “about the team member” posts on instagram**

**Oct 15, 2021**

**General Team Reorganization:**

**General team lead: Aarushi**

**Wet Lab team lead: Sophia**

**Wet Lab members: Sophia, Diya, Madison**

**Dry Lab team lead: Tanisha**

**Dry Lab team members: Tanisha, Maya, Aarushi**

**HP team lead: Ayana**

**HP team members: Ayana, Maya, Kaylin**

**October - present**

**Working on wiki and wiki requirements**

**Creating/planning out the video**

**Writing all of the content for the wiki**

**Gathering information about interviews and summarizing them**

**Reflecting on our teams’ achievements so far and thinking about steps we can take to take our project to the next level**

![](images/logo-full-colour-408px.png)

![](images/renert-smaller-logo.png)

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# Team:RHS-Calgary/Engineering

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Engineering Success

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* [Notebook](https://2021.igem.org/Team%3ARHS-Calgary/Notebook)
* Wet Lab

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* Human Practices

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* Team

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* [Home](https://2021.igem.org/Team%3ARHS-Calgary)
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* [Notebook](https://2021.igem.org/Team%3ARHS-Calgary/Notebook)
* Wet Lab

  + [Experiments](https://2021.igem.org/Team%3ARHS-Calgary/Experiments)
  + [Results](https://2021.igem.org/Team%3ARHS-Calgary/Results)
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* Human Practices

  + [Human Practices](https://2021.igem.org/Team%3ARHS-Calgary/Human_Practices)
* Team

  + [Collaboration](https://2021.igem.org/Team%3ARHS-Calgary/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3ARHS-Calgary/Attributions)
  + [Members](https://2021.igem.org/Team%3ARHS-Calgary/Team)

# Engineering Success

# iterative design in our project

In our experimentation thus far, there have been minimal occasions where we have encountered non-trivial obstances. However, one main instance in which we faced such scenarios was when we conducted our **third main experiment**. In this experiment, the system was seen to behave unexpectedly a number of times as it deviated from our initially proposed hypothesis. This experiment was conducted in two 24-well plates, with each plate containing two identical trials of each positive and negative control(This is shown in Figure 2, below). Our **initial hypothesis** was as follows: *Wells containing trials 1 through 7 are negative controls and will thus not yield any change in appearance; However, wells containing trials 8 and 9 are positive controls and will in fact express a visible colour change from yellow to purple.* While our team was confident that the above hypothesis would accurately represent our desired results, we faced some rather concerning results instead. We found that trials one through three, in both plates and in each duplicate trial, failed every time in that they expressed an obvious colour change from yellow to purple, though the **expected result** was for there to be no color change.

Immediately after observing these results, our wet lab team diligently began to work on creating yet another hypothesis as to why our negative controls may have failed. Through upholding a fundamental understanding of the reaction in play, we discussed that a colour change can only occur when components of the Working Reagent solution are exposed to Hydrogen Peroxide. Using various bioinformatic, biochemical, and biological research articles, we narrowed down the possibilities regarding where the possible source of contamination arose from, hence the creation of another hypothesis: *When alpha-D-Glucose undergoes oxidation by GOx, a reaction takes place that can produce a byproduct of **vaporous Hydrogen Peroxide**. Due to H2O2’s highly volatile nature, it can react with the components of the colorimetric assay kit in surrounding wells containing negative controls, henceforth producing false-positive results.*

It is important to note that the negative controls affected were immediately next to the wells containing the positive controls in the plate. This observation is what led us to the conclusion that trace amounts of gaseous Hydrogen Peroxide were being produced as a byproduct of the reaction, thus only contaminating the wells directly surrounding the positive controls. In order to approach the system which yielded non trivial results, we determined that the wells needed to be isolated from the surrounding environment and from each other. The Wet Lab team approached this by sealing each plate with a thin sheet of **parafilm wax**, allowing it to be airtight enough to **eliminate contamination** through H2O2 vapours. Throughout our experimentation and design process, we faced both **setbacks and successes**. Above is a crucial example of how both engineering success and failures impacted our project. While our high school did not heavily restrict access to our lab, we maximized our time when not in person by upholding weekly meetings in which we planned several forthcoming experimental designs, reviewed laboratory protocols, and developed potential educational lectures corresponding to our topic.

![](images/figure2proper1.png)

*Figure 2:* Chart demonstrating an experimental design that tests the maximum and minimum concentrations of glucose and all possible controls.

![](images/fixedresults31.png)

*Figure 3:* Data shows some successes and failures of negative controls when testing various dilutions of GOX and Glucose in 9 different trials, each containing 200 microlitres of W.R. All positive controls yielded successful results.

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# Team:RHS-Calgary/Experiments

Experiments

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* Human Practices

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* [Home](https://2021.igem.org/Team%3ARHS-Calgary)
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* [Notebook](https://2021.igem.org/Team%3ARHS-Calgary/Notebook)
* Wet Lab

  + [Experiments](https://2021.igem.org/Team%3ARHS-Calgary/Experiments)
  + [Results](https://2021.igem.org/Team%3ARHS-Calgary/Results)
  + [Engineering Success](https://2021.igem.org/Team%3ARHS-Calgary/Engineering)
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* Human Practices

  + [Human Practices](https://2021.igem.org/Team%3ARHS-Calgary/Human_Practices)
* Team

  + [Collaboration](https://2021.igem.org/Team%3ARHS-Calgary/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3ARHS-Calgary/Attributions)
  + [Members](https://2021.igem.org/Team%3ARHS-Calgary/Team)

# Experiments

# Experiment 1:

## Testing Hydrogen Peroxide with the Colorimetric Ass​​ay Kit

In the very first steps of our experimentation, we began by fabricating an outline to test each of the components of our project at their most fundamental levels. Prior to attempting to conduct any genetic modifications, or making our design more convoluted, to create our fully fleshed out desired outcome, we did extensive research to pin-point a reaction that best suits our required reactants and products. With this, we also had to ensure that we target an enzyme that is substrate specific for **Beta-D-Glucose**. Once our research had been completed, we discovered that our forthcoming experiments will be solely based on the following reaction: **B-D-Glucose + O2 → H₂O₂ + D-Glucono-1.5-lactone**, catalyzed in the presence of an enzyme, **Glucose Oxidase**. The H₂O₂ produced will then form a secondary biochemical reaction with a working reagent kit containing the dye, Xylenol orange. As mentioned, our first step was to ensure our purchased kit produces a sufficient reaction when exposed to hydrogen peroxide. With this, we created 3 different solutions of H₂O₂ combined with Working Reagent, in **different concentrations**. These solutions were composed of 0.03% H₂O₂, 0.3% of H₂O₂, and 0% H₂O₂ (which served as a control), respectively. This test was conducted in two, 6-well plates, with 1 trial of each of the variables in both plates.

# Experiment 2:

## Testing the production of hydrogen peroxide through GOx

After ensuring that the individual components of our kit yielded the results that we were seeking, we set out to incorporate our **enzyme** into our reaction to begin testing the **efficiency and success of the enzymatic production of H₂O₂**. As the enzyme we are currently using is manufactured as a commercial product, its packaging states that at a minimum, a concentration of **26 units/mL** is required to produce a reaction under highly specific conditions. Due to this, we were required to conduct various complex stoichiometric and mathematical calculations to determine the precise volume of **GOx** required to produce a reaction. In the calculations we ensured that we considered all factors that were at play. In such scenarios, mathematical and stoichiometric computations are extremely crucial in applications to synthetic biology, and they are methods frequently used in a majority of labs. In the end, we layed out a design that allowed us to have **varying concentrations** of GOx to test its efficiency and rate of activation energy required to produce a reaction. The varied amounts of GOx, however, mainly aided in creating dilutions that would produce **1 unit of GOx per 1 micromole (µmol) of solution**. Our final volumes of GOx in a **1unit/1µmol** sample were **85µL, 128µL, and 267µL**. We began this experiment by making two solutions of glucose, one with a higher concentration than the other, so we can begin to form a colour gradient that is indicative of the different amounts of glucose that could possibly be present in a cat's urine. We created a solution of **5000mg/L of glucose**, and a second solution containing **8000mg/L glucose**, both of which were required to be in a final volume, with the added GOx and Working Reagent, of **50mL**. Then, we proceeded by calculating the volume of working reagent required to produce the desired final volume of solution. The kit our team ordered for the reagent, from Thermo Scientific, contains a Working Reagent A and a Working Reagent B. In order for the product to function correctly, a ratio must be upheld between reagent A and reagent B, stated as **1µL:100µL**, respectively. With this, we were also required, by the instructions of the kit, to ensure a secondary ratio of 10 volumes of working reagent to 1 volume of sample, which we classified as **1mL W.R.:100µL** sample, after further stoichiometric calculations. Next, we prepared the working reagent solution according to Thermo Scientific's Kit Protocol, with **110µL** of Reagent A and **11000µL** of Reagent B. The last step in the process was to recalculate the volume of each of the three main components required to obtain a final volume of 0.05L in each well in 2, 6-well plates, which can be seen in **Figure 1** below.

![](https://2021.igem.org/File:T--RHS-Calgary--Figure1.png)

*Figure 1:* Chart demonstrating an experimental design set-up to test the reaction between GOx, W.R., and Glucose.

# Experiment 3:

## Testing the reaction between WR, GOx and Glucose, with multiple control trials

One of the main experimental designs our team has fabricated thus far allowed us to ask the principle question: can we detect high levels of glucose through a visible colour change with the use of **GOx** and a colorimetric peroxide assay? Having been able to say yes to this fundamental question allowed us to then research precise concentrations of glucose in the urine of diabetic cats, as provided to us by vet technician, **Ms. Emily Chorwood**. We conducted this experiment multiple times, each time in two 24-well plates with two trials of each variable in both plates. The purpose of this experiment was to produce a colour gradient that illustrates the concentration of glucose in a minimally diabetic cat's urine, to a severely diabetic cat's urine. This range of concentrations initially included, in order from minimally diabetic to severely diabetic, **[2.8M], [5.5M], [14M], [28M], and [55M]**, where ‘M’ refers to Moles/Litre. Once we completed performing the above experiment, we narrowed down our variables by simply using the lowest concentration of glucose ([2.8M]) and the highest concentration ([55M]), as we needed to solidify our proof of concept, ensuring that our negative controls do not change color. With a new goal in mind we asked ourselves: Can we prove that we can successfully trigger a colour change in our **positive controls** and not our **negative controls** when we combine our biological components of Glucose Stock Solution (5000mg/L Glucose combined with Synthetic Urine to create a [55mM] concentration of Glucose), Synthetic Urine, GOx and Working Reagent under certain conditions and in particular dilutions? To answer this, we set out to create an experimental design which we referred to in our processes several times, which can be seen below in Figure 2.

![](https://2021.igem.org/File:T--RHS-Calgary--Figure2.png)

*Figure 2:* Chart demonstrating an experimental design that tests the maximum and minimum concentrations of glucose and all possible controls.

# Experiment 4:

## Creating a Plasmid Construct and Protein Purification

# overview

Our team's ambition is to assemble our own plasmid and create a construct that can be contributed to iGEM, using the genome sequence of a GOx enzyme. We acquired a **His-tagged sequence of GOx** from independent research studies that an Isreali iGEM team previously conducted. The plasmid we construct will then be transformed into **BL21-E.Coli** bacterial cells. This particular strain of E.Coli is specifically engineered to efficiently express high levels of protein. After the transformation of BL21 with the constructed plasmid, the cells will be **induced**, and through the processes of transcription and translation, a GOx protein will be synthesized. Finally, we will conduct a **protein purification protocol** to isolate the GOx protein. Once we have genetically engineered the protein, we will also develop our own colorimetric peroxide assay kit.

# parts and resources

Initially, we intended to order **Wild-Type (WT) GOx**, a part fabricated by iGEM Manhattan College. Due to the fact that it no longer exists on the iGEM database, our team decided to attempt to re-create the part and hopefully re-enter it into the database for future use by other iGEM teams. With this, we intend to construct our plasmid, a process most commonly performed through in-vitro digestion of DNA fragments using **endonucleases (restriction enzymes)**, which are specific to certain restriction sites. The resulting fragments are then re-connected by a **ligase** enzyme. At that point, we will still have to manufacture the insert itself, as we were unable to order it, and we will then add it to our plasmid. As mentioned, our construct will have to be created in two separate components; the fundamental plasmid will be received from iGEM and the insert is from another plasmid, tailored to our desired endonucleases, retrieved from **Integrated DNA Technologies** (IDT). The process of removing one insert from a construct in order to place it into another is known as **subcloning**, otherwise known as the transfer of DNA from one vector to another to gain the ability and functionality to study or express a gene of interest. Throughout our processes, we navigated through many bioinformatics sources including  **Expasy**, an online bioinformatics resource operated by the **Swiss Institute of Bioinformatics** (SIB), providing us access to over 160 databases and software tools. Expasy enabled us to translate our nucleotide sequence to a polypeptide, otherwise known as a protein or sequence of amino acids.

# construct and purification

Our design for creating a highly specific plasmid construct begins with sending the sequence of GOx and our required restriction sites, **Xba1** and **Spe1**, to IDT to clone the protein sequence and manufacture it into a custom plasmid. The insert that will be a component of the IDT brand plasmid will contain the WT-GOx sequence with a **6x His-Tag site**, a T**EV site**, and two restriction sites. Once we receive the plasmid from IDT, the Xba1-GOx-Spe1 insert needs to be cleaved through the use of endonucleases using enzymes corresponding to specific restriction sites (Xba1 and Spe1). Restriction sites are only recognized by endonucleases that correspond to the **palindromic** sequence of DNA in the specific site. Succeeding this phase, we will take the **pSBC3-BBa\_K2238000** plasmid, from iGEM, and the restriction sites will be located. These sites are contained in a larger area called the **Multiple Cloning Site**, which contains a wide variety of sites for endonucleases to recognize.

Furthermore, we will then take the insert with the fabricated sticky ends and use a Ligase enzyme to connect it to the pSBC3 plasmid. By doing this step, the final plasmid construct is manufactured. Following the construction of our full plasmid, we will preliminarily have to transform it into **DH5-alpha competent** E.Coli bacterial cells. DH5a cells are genetically optimized for the increased and efficient replication of DNA, and this replication is to be done through a **QIAprep Miniprep procedure**. Then, another transformation will be completed in BL21 E.Coli cells, which are genetically modified and optimized to express high levels of protein. The expression of our target GOx protein will then be induced and we will grow high amounts of the final bacteria in our lab. These bacteria will then be **Lysed** and **purified**. The substance will undergo the process of **elution** in a **Nickel column**, which will separate our desired protein from all other cellular components. To ensure we have done this correctly, we will analyze the column using methods of **spectrophotometry**, which measures the amount of chemicals in a solution using a light beam that passes through samples.

## References

Text References

1.1.3.4: Glucose oxidase. BRENDA. (n.d.). Retrieved October 21, 2021, from https://www.brenda-enzymes.org/all\_enzymes.php?ecno=1.1.3.4&table=Temperature\_Stability.

Berg, J. M. (1970, January 1). The purification of proteins is an essential first step in understanding their function. Biochemistry. 5th edition. Retrieved October 21, 2021, from https://www.ncbi.nlm.nih.gov/books/NBK22410/.

The BioBricks Foundation:standards/technical/formats. OpenWetWare. (n.d.). Retrieved October 21, 2021, from https://openwetware.org/wiki/The\_BioBricks\_Foundation:Standards/Technical/Formats.

BL21 chemically competent E. Coli Cells. GoldBio. (n.d.). Retrieved October 21, 2021, from https://www.goldbio.com/product/14439/bl21-chemically-competent-e-coli-cells.

Chino, A., Watanabe, K., & Moriya, H. (n.d.). Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe. PLOS ONE. Retrieved October 21, 2021, from https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0009652.

Integrated DNA Technologies ǀ IDT. Integrated DNA Technologies. (n.d.). Retrieved October 21, 2021, from https://www.idtdna.com/pages.

IPE quick - sigmaaldrich.com. (n.d.). Retrieved October 21, 2021, from https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/208/756/ipe-quick-manual-ouc.pdf.

Lakna. (2018, April 6). How to read a plasmid map. Pediaa.Com. Retrieved October 21, 2021, from https://pediaa.com/how-to-read-a-plasmid-map/.

Nanodrop Lite interpretation of nucleic acid 260-280 ratios. (n.d.). Retrieved October 21, 2021, from https://tools.thermofisher.com/content/sfs/brochures/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf.

NE;, L. (n.d.). Protein purification: An overview. Methods in molecular biology (Clifton, N.J.). Retrieved October 21, 2021, from https://pubmed.ncbi.nlm.nih.gov/24648062/.

PBS - phosphate-buffered saline (10x) ph 7.4, RNase-free. Thermo Fisher Scientific - US. (n.d.). Retrieved October 21, 2021, from https://www.thermofisher.com/order/catalog/product/AM9624#/AM9624.

Primers/Catalog. (n.d.). Retrieved October 21, 2021, from http://parts.igem.org/Primers/Catalog.

SIB Swiss Institute of Bioinformatics. Expasy. (n.d.). Retrieved October 21, 2021, from https://www.expasy.org/.

Sticky ends. Oxford Reference. (n.d.). Retrieved October 21, 2021, from https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100532864.

Studier, F. W., & Moffatt, B. A. (2004, October 22). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology. Retrieved October 21, 2021, from https://www.sciencedirect.com/science/article/abs/pii/0022283686903852.

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# Team:RHS-Calgary/Results

Results

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# Results

# Experiment 1:

## Testing Hydrogen Peroxide with the Colorimetric Ass​​ay Kit

![](https://2021.igem.org/File:T--RHS-Calgary--resultsexperiment1.png)

*Figure 1:* Data demonstrates that Xylenol Orange from commercial colorimetric peroxide assay kit (in the Working Reagent solution) changes colour when exposed to Hydrogen Peroxide (H2O2).

The experiment demonstrated that the colorimetric assay kit does indeed change color when exposed to hydrogen peroxide. This is an essential part of our reaction as the color change is dependent entirely on the interaction between peroxide and the colorimetric assay kit. In this experiment, our hypothesis was proven true as the trials with 0.03% hydrogen peroxide changed color and those without hydrogen peroxide did not.

# Experiment 2:

## Testing the production of hydrogen peroxide through GOx

![](https://2021.igem.org/File:T--RHS-Calgary--resultsexperiment2.png)

*Figure 2:* Results shown demonstrate that a colour change occurs in high concentrations of Glucose and GOx when diluted with a small volume of W.R., and a colour-changing reaction partially occurs when the volume of GOx is nearly that of W.R.

# Experiment 3:

## Testing the reaction between WR, GOx and Glucose, with multiple control trials

![](https://2021.igem.org/File:T--RHS-Calgary--resultsexperiment3.png)

*Figure 3:* Data shows some successes and failures of negative controls when testing various dilutions of GOX and Glucose in 9 different trials, each containing 200 microlitres of W.R. All positive controls yielded successful results.

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