In order to identify potential issues and applications of our system, we engaged in direct dialogue with stakeholders, as per the Communication Strategies Guide (CSG). This approach allowed us to devise potential applications for our system, as well as correct design flaws such as the use of toxic pyocyanin as a redox-cycling molecule. This led us to repurpose our system with a safer molecule (phenazine methosulfate -PMS-), which also resulted in being a cheaper inducer molecule even when compared with broadly used inducers. We also identified that internal friction in teams is a common issue as proven to us by our experience as well as a survey that we conducted amongst 67 iGEM members from 14 other teams. To address this issue we developed our team communication app (LTAT) to help improve team communication both internally and in other teams.
Safety
Our genetic circuit is activated/deactivated by the redox state of the transcription factor (SoxR). SoxR oxidation is modulated by small redox molecules, such as pyocyanin. However, after Dr. Francesca Ceroni, a PI at Imperial who was pregnant at the time declined to meet us in the lab out of concern of toxic substances, we realized that toxicity is a huge factor in downstream implementation of our system. Pyocyanin is a toxin synthesised by the pathogen Pseudomonas aeruginosa and is implicated in its virulence (https://www.sciencedirect.com/science/article/pii/S0924857912002105?via%3Dihub). We also realized that using another cheaper redox molecule could not only make our system cheaper to use, but also replace inducer molecules such as IPTG due to their price. Using PMS which is a small redox molecule, we can activate a gene much like IPTG would with plac. Not only is PMS far cheaper than both pyocyanin and IPTG, it is also non-toxic and makes our system more applicable for real world applications.
Toxicity comparison between Pyocyanin and PMS
The 2012 OSHA Hazard Communication Standard ranks hazard ratings with the use of categories, with Category 0 being the lowest risk and Category 4 being the highest. With regards to toxicity, pyocyanin is a Category 4 substance and extreme care was taken during our wet lab to ensure our own safety and any contact with pyocyanin would warrant immediate medical attention, PMS on the other hand is a Category 0 substance and thus is far easier and safer to handle. OSHA pages on PMS (http://datasheets.scbt.com/sc-215700.pdf): OSHA pages on Pyocyanin (http://datasheets.scbt.com/sds/aghs/en/sc-205475.pdf).
Cost comparison between PMS and common inducer molecules
A cursory look at the costs of PMS, pyocyanin and common inducer molecules (such as IPTG) already reveal stark differences in costs per gram. When accounting for working concentrations, this difference is further magnified, with PMS being 268 or 2680 times cheaper than IPTG (depending on levels of IPTG used described by a NEB protocol provided here:https://www.neb.com/protocols/1/01/01/protein-expression-using-bl21de3-c2527) and 3584 times cheaper than pyocyanin. These costs are summarized in a table below. However costs only matter if it can be shown that PMS can have a similar fold induction to common inducer molecules such as IPTG and experimental results for fold induction suggesting that is the case can be found below.
Click here for experimental results
Environment
Biocontainment
A big socio-ethical issue with using genetically engineered organisms is the issue of biocontainment. We recognized this as an issue by talking to members of the public as well as from the socio-ethics discussion. These organisms should not be released where they could potentially cause ecological damage by outcompeting or harming native species. While some may debate the impact of this ecological damage, it would be easier to persuade governments and its people to use GMOs when proper biocontainment measures are in place. This is especially true for our project. By transcribing growth retardants or toxins, like gp2 and MazF respectively, we can control where our bacteria will live and thus add a layer of biocontainment.
Click here for experimental results
Fabric Bioprinter
In preparation for our art exhibition, we discussed the integration of science and art with a student from the RCA. She mentioned that in fashion, chemical pollution as a result of the usage of dyes is prominent. We realized that using bacteria to synthesize dyes could provide for an ecologically friendly solution. Moreover, with the ability to pattern using our electrode array, we can design simple prints using MelA which is a step in the right direction for the fashion industry. We have also succeeded in cloning the MelA gene into our construct design.
Click here for experimental results
Wellbeing
When we started our project, many of us had personal as well as interpersonal issues that threatened the viability of our project as well as our own well-being. We made it a point to reflect upon this experience and wondered if any other teams had similar issues to us. We surveyed 67 people from 13 different iGEM teams and developed a team-communication application called Let's Talk about It! as a aid for resolving these issues both for us and future iGEM teams. Most importantly, we have used the app to raise important issues with our PIs and supervisors and have these issues responded to as fast as possible. Knowing that this tool exists has made us more open about our issues and help each other communicate solutions for these issues. This has raised our productivity and made us more cooperative. More information on our Team-communication application can be found here
