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