Engineering
During the initial planning stage, our team decided on a project that focused on the detection of Antinuclear Antibodies, a common indicator of many autoimmune diseases. With goals of providing accessibility, we envisioned “ANA-Lyze this” to be a biosensor based rapid test that could be used in the comfort of one’s home. Below is the “ANA-Lyze This” logo created by team member, Tracy Wen.
Originally pitched by team members Steven Yang and Morteza Faraji, our team decided to take inspiration from the work done by Stewart Loh’s lab. In their 2022 paper, they created a switchable GFP biosensor that could change color upon binding to a ligand. We planned to adapt these principles to chromoproteins, which would offer the advantage of easy visual detection without specialized equipment. Chromoproteins are unique in that they change color upon binding to a target molecule, making them suitable for a visual, user-friendly diagnostic test. We had grand ambitions for "ANA-Lyze This," envisioning it as a groundbreaking tool, similar to that of a Covid-19 test, that could be easily used by individuals at home.
However, our journey took an unexpected turn when we presented our project idea to judges at the MindFuel's Tech Futures Challenge. Their feedback made us reconsider our project's direction. They pointed out several concerns, including the practicality of our project. It became evident that our initial project concept was not aligning with the desired global impact we sought.
In light of this feedback, our team decided to pivot away from the ANA detection sensor concept. We didn't abandon the innovative chromoprotein technology we had been exploring but instead refocused our efforts. We transformed our project into a versatile biosensor platform that could be adapted for various applications, Chromosense!
Our newly devised project aimed to harness the color-changing properties of chromoproteins in a way that would be both impactful and practical. After careful consideration, we decided to address a different challenge: Velvet disease, a severe health issue in captive marine fish caused by Amyloodinium dinoflagellate parasites. The parasite infiltrates a fish's slime coat and damages its cells, posing a significant problem for fish farmers and pet owners.
Using the constructs developed by the Uppsala iGEM team as our starting point, the focus of our biosensor is asPink and tsPurple, two remarkable chromoproteins that share the same chromophore sequence, CMYG. In order to create a switchable chromoprotein, the first step was to understand what makes asPink and tsPurple switch. What are the specific amino acids responsible for their distinct colors? Our journey takes us to amino acids I46, T94, and T95 in asPink. These particular amino acids drew our attention because they were not conserved between the two proteins and were predicted to interact with the chromophore in our structural models. Therefore, our strategy involves replacing these amino acids in asPink with their counterparts from tsPurple: T46, Q94, and I95. By doing so, we aim to pinpoint the essential amino acids that govern color change.
After site-directed mutagenesis, it was found that T94 and T95 were not essential for the colour differences. We have been unable to mutate I46 yet. Therefore, we need to go back to the amino acid sequences and investigate other differences. This will help identify how we can change asPink into tsPurple using strand displacement.
During the engineering cycle numerous challenges and unforeseen obstacles are encountered, underscoring the importance of project re-evaluation and adaptation of plans. Our experiences affirm that the initial phase, which involves defining the problem, is paramount and cannot be circumvented. A well-formed problem statement not only provides a clear direction but also serves as a goal throughout the development process. It has become evident that attempting to fit an existing technology to address a problem, without a thorough understanding and definition of the issue at hand, is a misaligned approach. Instead, the technology or solution developed should be tailored to meet the specific needs of the identified problem, ensuring that the following stages of the engineering cycle— from conceptualization to implementation— are effectively aligned toward a viable solution.
As discussed on our Project Description page, many steps are necessary to produce a working biosensor, which we can implement to further our project.
