iGEM 2019 — Team Concordia-Montreal

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Concordia-Montreal/Notebook

[![](https://static.igem.org/mediawiki/2019/b/b5/T--Concordia-Montreal--logoQ.png)](https://2019.igem.org/Team%3AConcordia-Montreal)

* Team ▾
  + [Team](/Team%3AConcordia-Montreal/Team)
  + [Collaborations](/Team%3AConcordia-Montreal/Collaborations)
  + [Attributions](/Team%3AConcordia-Montreal/Attributions)
* Project ▾
  + [Background](/Team%3AConcordia-Montreal/Background)
  + [Description](/Team%3AConcordia-Montreal/Description)
  + [Experiments](/Team%3AConcordia-Montreal/Experiments)
  + [Notebook](/Team%3AConcordia-Montreal/Notebook)
  + [Model](/Team%3AConcordia-Montreal/Model)
  + [Results](/Team%3AConcordia-Montreal/Results)
  + [Demonstrate](/Team%3AConcordia-Montreal/Demonstrate)
  + [Measurement](/Team%3AConcordia-Montreal/Measurement)
  + [Future Plans](/Team%3AConcordia-Montreal/Future-Plans)
  + [Supplementary](/Team%3AConcordia-Montreal/Supplementary)
* Parts ▾
  + [Basic](/Team%3AConcordia-Montreal/Basic_Part)
  + [Composite](/Team%3AConcordia-Montreal/Composite_Part)
  + [Part Collection](/Team%3AConcordia-Montreal/Part_Collection)
* [Safety](/Team%3AConcordia-Montreal/Safety)
* [Human Practices](/Team%3AConcordia-Montreal/Human_Practices)
* Medal Criteria ▾
  + [Bronze](/Team%3AConcordia-Montreal/Bronze)
  + [Silver](/Team%3AConcordia-Montreal/Silver)
  + [Gold](/Team%3AConcordia-Montreal/Gold)
  + [Special Awards](/Team%3AConcordia-Montreal/Hardware)
* [Judging Form](https://igem.org/2019_Judging_Form?id=2944)

Notebook

Notebook

* [1
  Biosensor](#Biosensor)
* [2
  Electrochemistry](#Electrochemistry)
* [3
  Electronic Device](#Device)
* [4
  Mobile Application](#Software)

**Contents:

1. Biosensor Notebook
2. Electrochemistry Notebook: Hydrogel and Redox materials and methods
3. Electrical Notebook
4. Software and Mobile Application Notebook**

## 1. Biosensor

Download the PDF:

[Notebook June/July](https://static.igem.org/mediawiki/2019/7/78/T--Concordia-Montreal--Notebook0607.pdf)

[Notebook August](https://static.igem.org/mediawiki/2019/e/ef/T--Concordia-Montreal--Notebook08.pdf)

[Notebook October](https://static.igem.org/mediawiki/2019/5/52/T--Concordia-Montreal--Notebook10.pdf)

[Notebook](https://static.igem.org/mediawiki/2019/7/76/T--Concordia-Montreal--GCRVP19lala.pdf)

## 2. Electrochemistry

 There are three different gels used: Chitosan, Agarose and a Cryogel

1. Cellular-functionalized Chitosan hydrogel

The first is the chitosan hydrogel which houses our engineered yeast cells and glucose for binding to GOx. Chitosan is biodegradable, biocompatible and regarded safe for human dietary use (Mohammed 2017). Chitosan is a biopolymer processed from chitin, which is found in crustacean shells such as shrimp. To prepare chitosan, insoluble chitin is processed commonly in acetic acid over heat, which causes deacytelation and renders it soluble. As it polymerizes, the gel is rendered a highly interconnected matrix of pores.
Chitosan medium molecular weight (448877, Sigma) was selected for initial experiments. Viscosity, or resistance to flow, is directly related to molecular weight. To ensure cellular material was retained without leakage as well as to provide viscosity high enough to retain cellular material in the chitosan gel, medium molecular weight was selected for initial experiments. The gel was prepared at 0.5 wt%/v 0.1M acetic acid. The low weight percent ensures large pore size for ample diffusion of molecules. Bovine serum albumin is cast with the cellular material into the gel to aid in undisrupted flow of proteins.

2. Buffered Agarose hydrogel

The agarose hydrogel lies against the skin, uptakes sweat and ISF and acts as a buffer against pH buildup and charge due to polarized molecules gathering at the skin.
Agar is a biopolymer generally obtained from red algae consisting of agaropectin and agarose. Agaropectin has more charged side groups and therefore can interact in unwanted ways with DNA or proteins. For this reason, agar is purified to agarose which is much more neutral and has a lower chance of interacting with biomolecules.
An agarose with a high electroendosmosis (EEO) value, or movement of liquid through a gel, is desired to optimize the speed of detection. We selected a medium EEO for molecular biology with an EEO >= 0.15 (A6877, Sigma) due to budget constraints for initial testing. The paper after which we modelled the system used Agarose type IV for molecular biology with an EEO >0.30 for which we plan to use in the future to optimize the system.
The agarose powder is prepared in a potassium phosphate buffer and heated until dissolution.

3. Drug delivery Cryogel

The cryogel was not made for this phase of the project. The pilocarpine drug delivery would be designed as per Kim & al. This hydrogel is a cryogel which is optimized for controlled pilocarpine drug delivery chosen for osmotic properties among others. When induced by a small current sent from the PCB via conductive inks, pilocarpine is repelled and delivered into the skin, inducing sweat and an electroosmotic flow of molecules which allows sampling of the ISF in addition to sweat.

References:
Conductive Inks

Silver/ Silver Chloride Ink (Ag/AgCl) and Carbon/ Graphite Paste (CP) were selected to carry current from the biosensor to the printed circuit board. Ag/AgCl ink is durable and a well-characterized reference electrode. CP is necessary to carry current from the Prussian Blue (PB) oxidation reaction to the Ag/AgCl ink. We chose to incorporate Prussian Blue into the carbon/ graphite paste by mortar and pestle with paraffin as a binder as per Moscone.

Insulator Ink
The insulator ink acts as a thermal barrier from the electrodes and PCB and the hydrogels housing cellular material.

Preparation

The electrodes were screen-printed by hand using a DIY screen-printing frame with mesh. The electrode pattern was printed on plastic and cut with an exacto knife into a stencil. Conductive inks used were Ag/AgCl Medical Grade Electrically Conductive Ink (117-23, Creative Materials, MA) with Thinner (102-03, Creative Materials, MA) until desired viscosity was attained. Carbon/ Graphite Paste (901969, Sigma) activated with Prussian Blue (PB), (234125, Sigma) acts as the working and counter electrode. The Prussian Blue electrodes are to be constructed and tested via cyclic voltammeter in iGEM Concordia futures.

Ag/AgCl ink was thinned to desired viscosity and screen-printed onto Silhouette temporary tattoo paper. It was then cured at 80\*C for 4min. Carbon/Graphite paste activated with Prussian Blue is screen-printed over the working and counter electrode. An insulator ink is silk-screened over all electrode areas to prevent heat or charge transfer.
Chitosan solution(o.5 wt%) in acetic acid was prepared. Yeast cells engineered for fentanyl detection are mixed with Bovine Serum Albumin (BSA), D-Glucose and chitosan hydrogel solution. The resulting mixture is cast onto the working Prussian Blue electrode. The same solution without cells is cast on the counter electrode. The agarose hydrogel containing buffer is prepared and silk-screened over the whole area.

References:

Mohammed, M., Syeda, J., Wasan, K., & Wasan, E. (2017). An Overview of Chitosan Nanoparticles and Its Application in Non-Parenteral Drug Delivery. Pharmaceutics, 9(4), 53. doi: 10.3390/pharmaceutics9040053

Kim, J., Sempionatto, J. R., Imani, S., Hartel, M. C., Barfidokht, A., Tang, G., … Wang, J. (2018). Simultaneous Monitoring of Sweat and Interstitial Fluid Using a Single Wearable Biosensor Platform. Advanced Science, 5(10), 1800880. doi: 10.1002/advs.201800880

M.Ahmed, E., Hydrogel products constitute a group of polymeric materials. (2013, July 18). Hydrogel: Preparation, characterization, and applications: A review. Retrieved July 14, 2019, from https://www.sciencedirect.com/science/article/pii/S2090123213000969?via=ihu

Khaled, Mustapha, & O, A. (2017, November 10). Immobilization of Glucose Oxidase in Anthracene-Based Semi-Conducting Polymer: Application on Glucose Biosensing. Retrieved July 12, 2019, from https://www.omicsonline.org/open-access/immobilization-of-glucose-oxidase-in-anthracenebased-semiconducting-polymer-application-on-glucose-biosensing-2155-9538-1000239-95278.html.

Oluwafemi, O. S., Kalarikkal, N., Thomas, S., & Songca, S. P. (2016, March 9). Biopolymers – Application in Nanoscience and Nanotechnology. Retrieved July 12, 2019, from https://www.intechopen.com/books/recent-advances-in-biopolymers/biopolymers-application-in-nanoscience-and-nanotechnology.
Silver-Silver Chloride Inks. Retrieved October 02, 2019, from http://www.creativematerials.com/products/ag-agcl-inks/.

Creative Materials
117-23 Medical Grade Electrically Conductive Ink, 102-03 Thinner

Sigma
D2O, DCl
Carbon/Graphite Screen printing paste- 901969
Iron (III) Ferrocyanide- 234125,
Glucose oxidase type X-S -G7141
Chitosan Medium Molecular Weight- 448877
Agarose Medium EEO for Molecular Biology- A6877
Hydrocortisone H-4001

Other
Potassium Diphosphate/ Monophosphate
D-Glucose
Silhouette Tattoo base paper (amazon.ca)

## 3. Electronic Device

*May 23, 2019 - May 24, 2019*- We defined the electronic device design specifications and requirements.
- We created a functional diagram of the electronic device.

*May 24, 2019 - May 27, 2019*- We performed a trade-off analysis to choose the electronic components that will meet the specifications and requirements.

*May 27, 2019 - June 8, 2019*- We designed and completed the circuit schematics of the design prototype.

*June 8, 2019 - June 20, 2019*- We designed and completed the printed circuit board layout of the design prototype.
- We constructed a breadboard circuit setup to test the electronic device's software.
- We developed a basic bluetooth software interface on the device.

*June 20, 2019 - July 1, 2019*- We developed a basic sensor software interface on the device.

*July 1, 2019 - July 5, 2019*- We assembled the design prototype circuit board.

*July 5, 2019 - July 11, 2019*- We troubleshooted and tested the design prototype circuit board.

*July 11, 2019 - July 13, 2019*- We designed and completed the product prototype circuit schematics.

*July 13, 2019 - July 18, 2019*- We designed and completed the product prototype printed circuit board layout.

*August 18, 2019 - August 21, 2019*- We revised the product prototype design and prepared it for manufacturing.
- We generated a 3D model of the product prototype to assist in the design.

*September 19, 2019 - September 20, 2019*- We assembled the product prototype circuit board.

*September 20, 2019 - September 30, 2019*- We troubleshooted and tested the product prototype circuit board.

*September 30, 2019 - October 20, 2019*- We updated and completed the microcontroller software to reflect changes in the design.

## 4. Mobile Application

*July 1, 2019 - July 11, 2019*- We brainstormed and defined ideas for the mobile application.

*July 11, 2019 - July 23, 2019*- Developed and completed the mobile application prototype and wireframe.

*July 23, 2019 - August 11, 2019*- We implemented the user status feature.

*August 11, 2019 - August 17, 2019*- We implemented the user account feature.

*August 17, 2019 - August 19, 2019*- We implemented the image processing feature.

*August 19, 2019 - August 30, 2019*- We implemented the data management feature.

*August 30, 2019 - September 8, 2019*- We implemented the help platform feature.

*September 8, 2019 - October 13, 2019*- We updated and completed the mobile application development.

#### Quantifen - iGEM Concordia 2019 Contact us at igem.concordia@gmail.com Follow us on social media! Instagram: igem\_concordia\_2019 Facebook page: @iGEMConcordia


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Concordia-Montreal/Experiments

[![](https://static.igem.org/mediawiki/2019/b/b5/T--Concordia-Montreal--logoQ.png)](https://2019.igem.org/Team%3AConcordia-Montreal)

* Team ▾
  + [Team](/Team%3AConcordia-Montreal/Team)
  + [Collaborations](/Team%3AConcordia-Montreal/Collaborations)
  + [Attributions](/Team%3AConcordia-Montreal/Attributions)
* Project ▾
  + [Background](/Team%3AConcordia-Montreal/Background)
  + [Description](/Team%3AConcordia-Montreal/Description)
  + [Experiments](/Team%3AConcordia-Montreal/Experiments)
  + [Notebook](/Team%3AConcordia-Montreal/Notebook)
  + [Model](/Team%3AConcordia-Montreal/Model)
  + [Results](/Team%3AConcordia-Montreal/Results)
  + [Demonstrate](/Team%3AConcordia-Montreal/Demonstrate)
  + [Measurement](/Team%3AConcordia-Montreal/Measurement)
  + [Future Plans](/Team%3AConcordia-Montreal/Future-Plans)
  + [Supplementary](/Team%3AConcordia-Montreal/Supplementary)
* Parts ▾
  + [Basic](/Team%3AConcordia-Montreal/Basic_Part)
  + [Composite](/Team%3AConcordia-Montreal/Composite_Part)
  + [Part Collection](/Team%3AConcordia-Montreal/Part_Collection)
* [Safety](/Team%3AConcordia-Montreal/Safety)
* [Human Practices](/Team%3AConcordia-Montreal/Human_Practices)
* Medal Criteria ▾
  + [Bronze](/Team%3AConcordia-Montreal/Bronze)
  + [Silver](/Team%3AConcordia-Montreal/Silver)
  + [Gold](/Team%3AConcordia-Montreal/Gold)
  + [Special Awards](/Team%3AConcordia-Montreal/Hardware)
* [Judging Form](https://igem.org/2019_Judging_Form?id=2944)

Experiments

Experiments

* [1
  Biosensor](#Biosensor)
* [2
  Electrochemistry](#Electrochemistry)
* [3
  Electronic Device](#Device)
* [4
  Mobile Application](#Software)

**Contents:

1. Biosensor: Brief Materials and Methods

2. Electrochemistry

3. Electronic Device

4. Mobile Application**

## 1. Biosensor: Brief Materials and Methods

The two plasmids used for our biosensor were assembled using Golden Gate assembly. DNA fragments of interest were amplified with specific sequences on both sides of the gene which contained restriction enzyme. In the presence of specific restriction enzymes, an overhang would be exposed on the gene of interest. These overhangs would then bind to complementary overhangs on the adjacent fragments, allowing for an ordered assembly of multiple fragments in one reaction. Type IIs restriction enzyme are used for this assembly, specifically BsaI and BsmBI. For our system, we used parts from the Moclokit to help assemble our vectors. Therefore, our overhangs were designed to be complement with those of the Moclokit (Lee, 2015). Saccharomyces cerevisiae was chosen to be our final host species was because the Moclokit is primarily constructed using yeast parts, therefore utilizing a yeast host allow our genes to be expressed efficiently.

The first step for our assembly was to assure that our genes of interest were codon optimized for a yeast system, which meant making sure that the amino acid sequences did not interact with any of the known yeast mechanisms. Afterwards, our genes of interest were flanked with specific sequences on the 5’ and 3’ ends. These sequences contain a primary and secondary set of overhangs that will be used for the Golden Gate assembly. Our genes of interest were either ordered with these specific sequences, or were amplified through polymerase PCR with the appropriate primers. Once we had our linear DNA fragment flanked by the correct sequences, it was inserted into an entry vector (PYTK001). DNA fragments can be inserted into this entry vector through the use of of a BsmbI digest, which exposes the primary complementary overhang. PYTK001 plasmids contain a green fluorescent protein (GFP) gene that are flanked by these BsmBI restriction sites, therefore in the presence of the restriction enzyme, the GFP gene would be removed and replaced with our genes. This design is useful for determining whether our colonies contain the correct vectors.

The potential assembly would then be transformed into a strain of E.coli (DH5ɑ). The transformant would be plated, and chosen through the presence/absence of GFP. Successful assembly is indicated by GFP positive colonies, however colony PCR is also performed to confirm whether the gene of interest was successfully inserted into the entry vector. This colony PCR would utilize primers that would bind on the PYTK001 plasmid just outside of the inserted sequence.

Once the gene of interest has been confirmed to be inserted into the entry vector, positive colonies would be innoculated overnight in LB media with appropriate antibiotic resistance. The plasmid would then be isolated from liquid inoculation through plasmid preparation. This isolated plasmid would then be ready to be used in the final Golden Gate assembly. This assembly would consist of assembling the gene of interest, antibiotic resistance gene, auxotrophic marker, promoter, terminator, assembly connectors and origin of replication for yeast and bacteria. Overall, eight plasmids with essential genes will be used in our Golden Gate assembly to construct our final vector. Plasmid 1 and plasmid 2 will be constructed using different auxotrophic markers, allowing for selection of specific colonies that have been successfully co-transformed into the same host cell. This final assembly would utilize BsaI restriction enzyme sites to expose the secondary complementary overhangs allowing for ordered assembly. Similarly to the entry vector, this final construct uses PYTK084 as its backbone, which contains a red fluorescent protein (RFP) gene flanked by BsaI restriction sites. In the presence of BsaI, the RFP gene is removed and replaced with the essential genes.

After, the potential plasmids constructs are transformed in E.coli and the cells are plated on an antibiotic specific media. The RFP negative colonies would then be selected, as they would indicate that the essential genes replaced the RFP gene and were successfully inserted within the vector. A secondary verification would be performed using colony PCR with specific primers that would amplify three of the potential inserted genes. Performing this experiment would verify that the plasmids have incorporated this gene of interest and have successfully assembled all of the genes in the correct order.

Both plasmids 1 and plasmid 2 will follow the same procedure to be assembled. Once they have successfully assembled and confirmed with colony PCR, they noculated overnight in LB media with appropriate antibiotic resistance and isolated using plasmid preparation. The verified vector construct will then be transformed into our yeast host, *Saccharomyces cerevisiae* (BY4742). After transformation, cells will be plated on a specific media that will allow viability of colonies only containing both plasmids, through auxotrophic selection. These cells would then be prepped to be inserted within our device.

![Two Plasmid System]( https://static.igem.org/mediawiki/2019/7/7c/T--Concordia-Montreal--Biosensor1.png)
**Figure 1. Genetic construct of the biosensor with a two-plasmid design.** Here, a first plasmid (P1) encodes a transcription factor (GAL4-FEN-VP16 or GAL4-GCR-VP16) which activates the inducible GAL1 promoter on the second plasmid (P2) upon ligand binding. The latter transcribes the reporter gene, either the enzyme glucose oxidase (GOx) or the chromoprotein amilCP to give a signal processable by our Quantifen app.

![In Yeast]( https://static.igem.org/mediawiki/2019/6/61/T--Concordia-Montreal--Biosensor_2.png)
**Figure 2. Complete overview of the biosensor, post-transformation into Saccharomyces cerevisiae.** Shown above is a single yeast cell transformed with both plasmids so that constitutive transcription of P1 induces transcription of the reporter gene on P2 when ligand (such as fentanyl or other small molecule with its respective receptor) binds the LBD.

## 2. Electrochemistry

\*Yeast viability in Hydrogel (ongoing)
Yeast viability is tested in the chitosan gel via dehydration and cryofreezing. The cells are prepared as a liquid culture, mixed by pipette with chitosan solution and set into well plates. Dehydration is done at 60 degrees Celsius for 30min. Cryofreezing is also performed. For both conditions, samples are prepared in and out of the gel.
The samples are rehydrated in YPD and in artificial sweat preparation (a solution at a lower pH). Testing is done with FDA/PI. Controls are prepared and recorded with fluorescent microscopy. Viable cells are counted to determine viability.

\*Fluorescence After Photo Bleaching to determine diffusion coefficient of chromoprotein in chitosan hydrogel (ongoing)
AmilCP is from the family of fluorescent proteins and therefore has a similar molecular weight and structure. We hence can use fluorescent protein methods to determine amilCP diffusion in the hydrogel.
E. coli transformed with Red Fluorescent Protein (RFP) are prepared in liquid culture then lysed, keeping the supernatant. The supernatant is pipetted into chitosan solution and set on a micro slide. The sample is bombarded with a laser near the excitation wavelength of RFP (~580nm) to a point of photo-bleaching. This results in an area of the sample which does not fluoresce, while the rest of the sample will. Through Brownian diffusion, the RFP proteins move back into the bleached area. The diffusion coefficient for RFP proteins can then be determined by consecutive image capture and appropriate calculations derived from Fick's Law.

\*Cyclic voltammeter to determine current created by GOx in and out of hydrogel layers (ongoing)
To determine the amount of charge created by the electrochemical transfer between hydrogen peroxide, a product of the reaction of glucose oxidase with glucose, and Prussian Blue activated electrodes.
Screen-printed Ag/AgCl electrodes activated with Prussian Blue electrocatalyst are tested. Varying concentrations of glucose are tested with a set amount of glucose oxidase. Then glucose oxidase concentrations are varied while glucose stays the same. Cyclic voltammetry is conducted to measure the current produced by the electrochemical transfer of electrons.

## 3. Electronic Device

### Schematics and Layout Debugging on KiCAD

During the design process, the schematics and layout design are constantly being revised. The revision process relies on two methods: breadboard testing and software debugging.

Breadboard testing uses through-hole versions of the various circuit components to build a circuit on a breadboard. Testing is performed by individually testing the functionality of each integrated circuit and verifying the correctness of their application circuits. Then, the electronic device’s circuit is constructed and the operation of each integrated circuit is verified in the context of the entire circuit. The breadboard setup is then used to develop and test the microcontroller’s software.

![](https://static.igem.org/mediawiki/2019/8/8f/T--Concordia-Montreal--BreadboardSetup.jpg)

**Figure 1:** Breadboard setup to develop the sensor interface

Software debugging implies using KiCAD to debug the circuit. KiCAD allows the designer to verify electrical rules following the table in Figure 2.

The electrical rules checker is used in the circuit schematics. To debug the printed circuit board layout, KiCAD has a design rule checker function which allows the designer to check for unconnected traces, components and traces clearance and constraints.

![](https://static.igem.org/mediawiki/2019/d/d1/T--Concordia-Montreal--ElectricalRuleChecker.PNG)

**Figure 2:** The electrical design rule checker on KiCAD

### Design Prototype

The design prototype corresponds to the first version of the electronic device which purpose is to test and troubleshoot the operation of the electronic device. The design prototype is created on a single board to reduce cost.

Testing of the design prototype involves verifying proper voltage distribution and functionality of the microcontroller software such as data collection and transmission.The testing procedure for the design prototype is as follows:

1. Evaluate the voltage at +BATT, +2V85, +1V8 terminals relative to GND for every component.
2. Upload the microcontroller software onto the microcontroller.
3. Transmit dummy data to the mobile application.
4. Collect data from the sensors and adjust sensor configurations.
5. Evaluate voltage at the iontophoresis control pin on the microcontroller.

![](https://static.igem.org/mediawiki/2019/4/41/T--Concordia-Montreal--DesignPrototypeTest.PNG)

**Figure 3:** Testing for the voltage distribution on the design prototype.

Once these steps have been performed on the design prototype and the issues found has been troubleshooted, then the design for the product prototype can be initiated.

### Product Prototype

The second product corresponds to the second version of the electronic device which purpose is to demonstrate the viability of the product design and test and troubleshoot the updated electronic device based on changes made on the design prototype. The product prototype follows the two-board design.

Testing of the product prototype involves verifying proper voltage distribution, functionality of the microcontroller software and structural viability of the electronic device. The testing procedure for the product prototype is as follows:

1. Evaluate the voltage at +BATT, +2V85, +1V8 terminals relative to GND for every component.
2. Upload the microcontroller software onto the microcontroller.
3. Transmit dummy data to the mobile application.
4. Collect data from the sensors and adjust sensor configurations.
5. Evaluate voltage at the iontophoresis control pin on the microcontroller.
6. Apply compression to the assembled electronic device and observe if it bends.

![](https://static.igem.org/mediawiki/2019/e/ef/T--Concordia-Montreal--ProductPrototypeTesting.PNG)

**Figure 4:** Testing for the voltage distribution on the product prototype.

## 4. Mobile Application

### Functional Testing of the Mobile Application

Testing a mobile application is done at every stage of its development. Every new function is tested against a variety of conditions. For instance, testing the email address text field in the LogInActivity activity involves trying string of characters that do not correspond to the format of an email address, trying an empty field, trying with capital letters, omitting the “.com”, omitting the “@” symbol, trying without any characters before the “@”, trying without any character after the “@”, trying without any character between the “@” and the “.com”, trying without any character before “.com”.

Describing the entire testing procedure of the entire mobile application is hardly feasible. However, a few general guidelines are followed when testing the mobile application. First, we ask the question: “What do we want this function/feature/field to do?”. From this question, we test and determine whether the said task is performed within the requirements. Then, we ask the question: “What do we not want this function/feature/field to do?”. From this question, we test a range of inputs that we know should not and do not want to have the same effect as an input that is desirable. For instance, a proper email address for an email address text field would be: “john@doe.com”. We test this input and see if the mobile application accepts it. If it does, we pass the first question. Then, we test something that we do not want to work with the text field such as “123456” which is not an email address. If the mobile application rejects this input then we pass the second question.

A third question to be asked is: “How do I make the mobile application crash?”. To answer this question a range of inputs is applied to the mobile application and we observe if some combination of inputs will cause the mobile application to shut down or freeze.

However, the three questions are not enough to determine the quality of an application. There needs to be user feedback on the mobile application. The user can tell the developer. for instance, that the mobile application takes too long to load or that the navigation is too complex. Therefore, the mobile application will require beta testing from a significant pool of people. The current stage of the application does not allow such widespread beta testing yet. Only feedback from the team and mentors are being used at the moment.

### Integration Testing with the Electronic Device

Integration testing with the electronic device subsystem is an important aspect of this project. It ensures that a proper connection between the electronic device subsystem and the mobile application subsystem can be established. It also verifies that data can be transferred between the two subsystems. The testing involves connection and data testing as well as distance testing to verify the maximum range at which a proper signal can be obtained.

The other elements of the testing process are aimed at determining failure scenarios and providing insight into how to solve them. Among these are a shutdown of the electronic device subsystem, a shutdown of the mobile phone an unstable connection, an unresponsive mobile application.

#### Quantifen - iGEM Concordia 2019 Contact us at igem.concordia@gmail.com Follow us on social media! Instagram: igem\_concordia\_2019 Facebook page: @iGEMConcordia

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Concordia-Montreal/Results

[![](https://static.igem.org/mediawiki/2019/b/b5/T--Concordia-Montreal--logoQ.png)](https://2019.igem.org/Team%3AConcordia-Montreal)

* Team ▾
  + [Team](/Team%3AConcordia-Montreal/Team)
  + [Collaborations](/Team%3AConcordia-Montreal/Collaborations)
  + [Attributions](/Team%3AConcordia-Montreal/Attributions)
* Project ▾
  + [Background](/Team%3AConcordia-Montreal/Background)
  + [Description](/Team%3AConcordia-Montreal/Description)
  + [Experiments](/Team%3AConcordia-Montreal/Experiments)
  + [Notebook](/Team%3AConcordia-Montreal/Notebook)
  + [Model](/Team%3AConcordia-Montreal/Model)
  + [Results](/Team%3AConcordia-Montreal/Results)
  + [Demonstrate](/Team%3AConcordia-Montreal/Demonstrate)
  + [Measurement](/Team%3AConcordia-Montreal/Measurement)
  + [Future Plans](/Team%3AConcordia-Montreal/Future-Plans)
  + [Supplementary](/Team%3AConcordia-Montreal/Supplementary)
* Parts ▾
  + [Basic](/Team%3AConcordia-Montreal/Basic_Part)
  + [Composite](/Team%3AConcordia-Montreal/Composite_Part)
  + [Part Collection](/Team%3AConcordia-Montreal/Part_Collection)
* [Safety](/Team%3AConcordia-Montreal/Safety)
* [Human Practices](/Team%3AConcordia-Montreal/Human_Practices)
* Medal Criteria ▾
  + [Bronze](/Team%3AConcordia-Montreal/Bronze)
  + [Silver](/Team%3AConcordia-Montreal/Silver)
  + [Gold](/Team%3AConcordia-Montreal/Gold)
  + [Special Awards](/Team%3AConcordia-Montreal/Hardware)
* [Judging Form](https://igem.org/2019_Judging_Form?id=2944)

Results

Results

* [1
  Construction of Plasmids](#Construction of Plasmids)
* [2
  Characterization of BBa\_K2944003](#Characterization of BBa_K2944003)
* [3
  Protein Purification and Growth Curve](#Protein Purification and Growth Curve)
* [4
  Electronic Device](#Electronic Device)
[5
Mobile Application](#Mobile Application)

**Contents:

1. Construction of Plasmids
2. Characterization of BBa\_K2944003
3. Protein Purification and Growth Curve of BBA\_K1692032
4. Electronic Device
5. Mobile Application**

## 1. Plasmid Construction

The goal of the biosensor section of this project was to construct two plasmids that would be responsible for detecting a small molecule (Plasmid 1) and produce signal upon detection of that small molecule (Plasmid 2). Throughout this process, multiple steps were required to ensure that our construction would be functional.

The primary step was to successfully amplify our genes of interest with the right sequences to allow for golden gate assembly. For Plasmid 2, the genes of interest that we were working with were Glucose oxidase (GOx) and the Chromoprotein AmilCP.

Due to an inability to order the entire GOx gene (because it was too large), we had to order it as two separate parts. Therefore, each of the two GOx part (designated as GOx part 1 and GOx part 2) were required to be amplified separately before they could be inserted into the entry vector. Specific primers were designed to assemble the two without interfering with the genetic sequence. As seen in Figure 3, both parts were successfully amplified with the appropriate primers, with both sequence being present near the 1.0 kb line of the ladder.

![PCR amp of GOx](https://static.igem.org/mediawiki/2019/1/15/T--Concordia-Montreal--Resultsfig3.png)
**Figure 1: PCR amplification of GOx part 1 and GOx part 2 with overhangs.**  Lane 1; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2; GOx part 1 (~1.00kb), Lane 3; GOx part 2 (~0.98 kb).

After confirming that both parts were successfully amplified, they were used in a Golden Gate reaction using the entry vector PYTK001. Assembly reaction was performed and plated (Fig.4). GFP-negative colonies were selected and a colony PCR was performed to confirm the vector assembly as seen in Fig. 5. Gel confirmed entry vector with bands present near the 2.0kb line of the ladder, which is the same size as the complete GOx sequence that was amplified.

![GOx-001 vector]( https://static.igem.org/mediawiki/2019/b/b1/T--Concordia-Montreal--Resultsfig4.png)
**Figure 2: GOx-001 vector.** Plate demonstrates GOx part 1 and GOx part 2 inserted in PYTK001 entry vector and transformed in E.coli.

![GOx-001 cPCR](https://static.igem.org/mediawiki/2019/1/1f/T--Concordia-Montreal--Resultsfig5.png)
**Figure 3: GOx-001 cPCR.** Lane 1, 22, 23, 31; GeneRuler 1 kb Plus DNA Ladder (Thermo fisher), Lane 2-21, 24-28; GOx-001 (~2.021kb)

After the successful incorporation into the entry vector, the GOx plasmid was ready for a final Golden Gate assembly with the other essential genes. The assembly was verified with cPCR as seen in Fig.6. The cPCR performed with the assemble vector (dubbed CEN6LEU-GOx because of the presence of the CEN6/ARS4 origin of replication, LEU3 auxotrophic marker and Glucose oxidase) utilized a primer that would amplify the promoter (TDH3), GOx and terminator (PGK1). Demonstrating that these three genes were present verified that the vector was assembled in the correct order and contained the gene of interest.

![GOx-001 cPCR](https://static.igem.org/mediawiki/2019/d/d2/T--Concordia-Montreal--Resultsfig6.png
)
**Figure 4: CEN6LEU-GOx cPCR.** Lane 1; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2-4; CEN6LEU-GOx amplification (~2.96kb)

For our AmilCP, the gene was extracted from one of the parts provided by the iGEM kit, BBa\_K1692032. Once transformed and plasmid prepped, specific primers were used to amplify the amilCP gene and provide the appropriate overhangs. Figure 7 shows a gradient PCR was performed for the specific sequence. However, due to the presence of multiple bands, the gene needed to be gel extracted. Once that was complete, it was ready to be assembled into the entry vector. Entry vector was constructed, with selection of GFP negative colonies (Fig 8), and a cPCR was performed to confirm results as seen in Fig 9. The primers amplified the insert within the entry vector, which was approximately 712 bp. Lane 3 demonstrated a successful assembly and a plasmid preparation was performed to isolate the vector. Unlike the previous insert, the final vector was unable to be completed due to lack of time and troubleshooting.

![AmilCP](https://static.igem.org/mediawiki/2019/e/ed/T--Concordia-Montreal--Resultsfig7.png
)
**Figure 5: AmilCP amplifciation with overhangs.** Lane 1 and 10; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2-9; amilCP (~7.0kb)

![AmilCP](https://static.igem.org/mediawiki/2019/5/57/T--Concordia-Montreal--Resultsfig8.png)
**Figure 6: AmilCP-001 vector.** Plate demonstrates AmilCP inserted in PYTK001 entry vector and transformed in E.coli.

![AmilCP](https://static.igem.org/mediawiki/2019/2/28/T--Concordia-Montreal--Resultsfig9.png)
**Figure 7: AmilCP-001 cPCR.**Lane 1; Quick-Load® Purple 1 kb Plus DNA Ladder (New England Biolab), Lane 2-7; AmilCP (~7.0kb)

As for plasmid 1, the gene of interest that was meant to be assembled was GGV (GAL4-GCR-VP16). The gene of interest was ordered, however due to lack of time, the plasmid was not able to be completed.

## 2

**Characterization by Mass Spectrometry of [Part:BBa\_K2944003](http://parts.igem.org/Part%3ABBa_K2944003).**
![MassSpec]( https://static.igem.org/mediawiki/2019/2/27/T--Concordia-Montreal--MassSPEresults.png)
[Preparation of Standard Curve.pdf](https://static.igem.org/mediawiki/2019/8/8f/T--Concordia-Montreal--GoxCharacterization.pdf)

[Preparation of Standard Curve Continued.pdf](https://static.igem.org/mediawiki/2019/6/6b/T--COncordia-Montreal--massspect.pdf)

[Preparation of Standard Curve Continued.pdf](https://static.igem.org/mediawiki/2019/6/6b/T--COncordia-Montreal--massspect.pdf)

Results:
Image built using Prism 6.

There are two superimposed traces: gluconolactone standard and yeast supernatant mixed 50/50 with 40 g/L glucose. The traces are of extracted ion 195.0505 (exact [M-H]- of gluconate. The gluconolactone standard is predominately gluconate upon resuspension in water.

The mass spectrum in the inset is the total ion count of the standard at 0.5 mins between the indicated m/z range. The two masses which are not the exact mass of gluconate are also part of the standard. Their identities are confirmed using the online database mzCloud. Annotated in the figure is we believe these masses correspond to based on the papers referenced below.

Characterization done by Matthew Tiranardi.
Thankyou Lauren Narcross for her technical assistance!
References:

Alieva, N. O., Konzen, K. A., Field, S. F., Meleshkevitch, E. A., Hunt, M. E., Beltran-Ramirez, V., … Matz, M. V. (2008). Diversity and evolution of coral fluorescent proteins. PLoS ONE, 3(7). https://doi.org/10.1371/journal.pone.0002680

Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase - An overview. Biotechnology Advances. https://doi.org/10.1016/j.biotechadv.2009.04.003

Lee, M., DeLoache, W., Cervantes, B., & Dueber, J. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975-986. doi: 10.1021/sb500366v

Taylor, V. F., March, R. E., Longerich, H. P., & Stadey, C. J. (2005). A mass spectrometric study of glucose, sucrose, and fructose using an inductively coupled plasma and electrospray ionization. International Journal of Mass Spectrometry, 243(1), 71–84. doi: 10.1016/j.ijms.2005.01.001

Zhang, Z., Gibson, P., Clark, S. B., Tian, G., Zanonato, P. L., & Rao, L. (2007). Lactonization and Protonation of Gluconic Acid: A Thermodynamic and Kinetic Study by Potentiometry, NMR and ESI-MS. Journal of Solution Chemistry, 36(10), 1187–1200. doi: 10.1007/s10953-007-9182-x

## 3. Protein Purification and Growth Curve

[See more here: Part:BBa\_K1692032](http://parts.igem.org/Part%3ABBa_K1692032)
![Circular Dischorism]( https://static.igem.org/mediawiki/parts/8/83/T--CONCORDIA-MONTREAL--CD_amilCP.png)

**Figure** A circular dichroism spectrum of amilCP indicates that the secondary structure is predominantly composed of β-sheets as indicated by the single minimum around 217nm and the maximum above 195nm. The minimal broadening towards 210nm and the second maximum at 190nm suggests that there are discrete α-helix structures in the proteins but are not a significant feature of this protein. The impact of the β-sheets on the ellipticity of circularly polarized light prevents any smaller features from being characterized. Furthermore, a negative of polarized light followed by a positive rotation of polarized light suggests that the β-sheets are arranged in an anti-parallel fashion throughout the protein.

## 4. Electronic Device

The objective of the electronic device is to serve as an interface between the biosensor/electrochemistry patch and the user. This objective has been met as all the components on the device are functioning nominally. The electronic device is thus able to produce an iontophoretic current, measure a low current input, measure temperature and transmit the data to an external Bluetooth device. The current design of the electronic device is the product prototype which corresponds to the second iteration of the design. It is shown in Figure 1 and 2.

![Size Comparison of the Product Prototype](https://static.igem.org/mediawiki/2019/4/4a/T--Concordia-Montreal--Electronic_Product_Prototype.jpg)

**Figure 1:** The product prototype's two boards compared to a Canadian quarter.

![Top View of the Electronic Device](https://static.igem.org/mediawiki/2019/c/c1/T--Concordia-Montreal--ElectronicTopView.jpg)
![Bottom View of the Electronic Device](https://static.igem.org/mediawiki/2019/d/de/T--Concordia-Montreal--ElectronicBottomView.jpg)

**Figure 2:** (a) Top view of the electronic device. (b) Bottom view of the electronic device.

## 5. Mobile Application

The objective of the mobile application is to provide an intuitive interface for the user who will be using our product This objective has been met as both front-end and back-end interfaces of the application are working. The mobile application can thus receive data from the electronic device, inform the user of their current condition, refer them to the nearest help locations. Some the screens of the mobile application are shown in Figure 3 and 4.

![Log and Join Activity](https://static.igem.org/mediawiki/2019/a/a4/T--Concordia-Montreal--Screen02_LogJoin.png)
![Sign Up Activity](https://static.igem.org/mediawiki/2019/b/bb/T--Concordia-Montreal--Screen03_SignUp.png)
![Log In Activity](https://static.igem.org/mediawiki/2019/7/70/T--Concordia-Montreal--Screen04_LogIn.png)
![Status Activity](https://static.igem.org/mediawiki/2019/e/e0/T--Concordia-Montreal--Screen01_Status.png)
![Emergency Activity](https://static.igem.org/mediawiki/2019/2/2b/T--Concordia-Montreal--Screen10_EmergencyContact.png)

**Figure 3:** (a) Log and join activity, (b) Sign up activity, (c) Log in activity, (d) Status activity, (e) Emergency activity.

![Camera Menu Activity](https://static.igem.org/mediawiki/2019/2/28/T--Concordia-Montreal--Screen05_CameraMenu.png)
![Image Analysis Activity](https://static.igem.org/mediawiki/2019/e/e5/T--Concordia-Montreal--Screen06_ImageAnalysis.png)
![Learn Menu Activity](https://static.igem.org/mediawiki/2019/2/24/T--Concordia-Montreal--Screen07_LearnMenu.png)
![Help Map Activity](https://static.igem.org/mediawiki/2019/2/20/T--Concordia-Montreal--Screen08_HelpCentersMap.png)
![Account Activity](https://static.igem.org/mediawiki/2019/f/f5/T--Concordia-Montreal--Screen09_Account.png)

**Figure 3:** (a) Camera menu activity, (b) Image analysis activity, (c) Learn menu activity, (d) Help center map activity, (e) Account activity.

#### Quantifen - iGEM Concordia 2019 Contact us at igem.concordia@gmail.com Follow us on social media! Instagram: igem\_concordia\_2019 Facebook page: @iGEMConcordia