Throughout our journey, we developed new tools and protocols, and had amazing discussions with other team members, advisors and experts. We are happy to share all the parts we created and the knowledge we acquired to pave the way for future iGEM teams!
Our final constructs were designed as plasmids composed of various parts, that respond to reactive oxygen species (ROS) by either emitting fluorescence or producing a specific enzyme. This functionality was achieved by coupling ROS-sensitive transcription factors to the expression of target proteins, creating a feedback loop adapting the system's sensitivity to ROS levels.
For this purpose, we created some new parts such as mutated promoter sequences, and combined them with known parts in different combinations to produce our final organisms.
An exhaustive list of the basic and composite parts we generated can be found here in the parts registry, as our contribution. These parts can serve as valuable tools for future research and development in ROS detection, sensing and related applications.
Months of work in the wet lab taught us that patience, perseverance, and optimism are essential ingredients for successful cloning, along with what sometimes feels like a touch of mystical luck. However, beyond the unpredictability, we also gained valuable practical insights. These tips are easy to implement and could help future teams navigate the complexities of cloning with more confidence. By sharing our solutions, we hope to provide others with the tools and strategies needed to tackle the challenges of the cloning world. We saw a lot of success with "speedier" transformation protocols, which saved us time in the lab. Find out more in our Protocols page.
Dry lab methods played an important role in predicting which nucleotides of the promoter were most involved in the binding interactions of our transcription factors. By using computational modeling and simulations, we were able to identify key nucleotides and explore how exchanging them could reduce the sensitivity of our system. The impact of mutations in transcription factor binding sites on DNA binding affinity was analyzed by a PSSM-based model was used to compute desensitized promoter sequences. These were ultimately synthesized and characterized in the wet lab. This predictive approach allowed us to fine-tune the behavior of the transcription factors, optimizing their performance for our specific needs.
We also developed a cell model based on ODEs, that illustrates how the lower binding affinity of a transcription factor to the promoter region changes the sensitivity threshold of the promoter and its protein expression.
The steps of our process are detailed on the Model page, and the code is available here in the iGEM Software repository. Our results are fully reproducible using the published code.
Through our discussions with experts as part of the integrated human practices part of the project, we gained valuable insights into IBD, fireblight and ROS sensing in general. For future teams working in these areas, it would be useful to quickly see current research trends and see which gaps still exist that their project may fill. We’ve compiled key takeaways from our interviews, providing condensed and actionable knowledge that can shorten the ideation and design process for teams interested in tackling any of the topics we propose to tackle in our project. Find out more in the Human Practices page!
In addition, we have put considerable thought into potential biocontainment measures that could be attempted in various bacterial chassis organisms that we could see applied in a real-life battle against ROS bursts. Find out more in the Safety page.