Experimental Validation
Our project focuses on developing biosensors using E. coli promoters to detect significant molecules in fields of healthcare, environmental monitoring, and biotechnology. We started off by identifying ten relevant molecules to test with each of the 2000 promoters in the E. coli library. We then identified which promoter-molecule pairs exhibited the highest fluorescence response in order to create a reliable basis for biosensor development. Our initial screenings provided valuable insights into how E. coli promoters respond to different molecules, but we noticed discrepancies when the promoter was titrated with increasing concentrations of its respective molecule. We expected the fluorescence to increase with a greater molecule concentration, but the experimental trends differed and the initial screening fluorescence was often much higher than that of the titration. To better understand these inconsistencies, the next step would be to conduct thorough validations with each molecule. We repeated the titration several times, changing a different variable in each trial to locate the source of the issue. For example, we regrew promoters with low cell growth, altered how long the strains were left in the incubator before use, repeated with uncontaminated media, and reran after fixing any mislabeled promoters. After each experiment, the data was analyzed, the experiments were revised, and further testing was conducted. Titrations for each molecule-promoter pairing were done in three replicates to ensure accuracy of the results. The initial screening round was also redone on some promoter plates several weeks later to confirm that these results were replicable. By resolving these issues, we can strengthen the reliability of our biosensor designs and move forward with its actual implementation.
Implementation
Once our promoter-molecule pairings have been validated, our findings can be leveraged to create functional biosensors for a range of practical applications. For example, we noticed strong promoter responses to pollutants like carbaryl, a widely-used carbamate insecticide [1], and perfluorooctane sulfonate (PFOS), a organic pollutant used in stain-resistant and non-stick products [2]. Biosensors designed to detect these molecules could be integrated into environmental monitoring systems to assess contamination levels in water and soil, providing real-time detection of sub-lethal concentrations and enabling quick remediation efforts. Additionally, our research identified a responsive promoter for lovastatin [3], a statin drug used to lower cholesterol by inhibiting the enzyme HMG-CoA reductase. In pharmaceutical manufacturing, lovastatin-responsive biosensors could be used for in-line monitoring of fermentation processes, ensuring accurate dosing and maintaining quality control during production. Further applications can include detecting quorum-sensing molecules like butanoyl-homoserine lactone, that can enable the monitoring of microbial communities and their communication processes. By focusing on these specific molecules, scientists can tailor biosensors to meet the critical needs in environmental safety, public health, and industrial biotechnology. Our interviews with stakeholders, including farmers, researchers, and synthetic biologists, provided perspective on biosensors implementation and potential limitations. For example, Jenny, a fourth-generation almond farmer, discussed the importance of accurate pesticide monitoring. She emphasized the need for affordable, efficient tools that can measure pesticide degradation to minimize environmental harm while maintaining crop health. However, there are high costs and other regulations involved in pesticide use, suggesting that biosensors should be cost-effective and comply with regulatory standards to be useful on the ground. In parallel, both synthetic biologists from the Badran Lab and Yiwen Jan, founder of a bioreagent company, talked about technical concerns. The Badran Lab team emphasized the need to optimize sensitivity and dynamic range of biosensors. They said that high cooperativity in biosensors could make accurate concentration measurements complicated and suggested that reducing cooperativity while maintaining sensitivity would be key to practical application. Yiwen Jan echoed similar concerns about the viability of biosensors in real-world conditions, questioning whether bacteria in our systems could even survive outside the lab and if they would even remain functional over time. Addressing these limitations— from regulatory and cost concerns in farming to technical challenges in sensitivity and durability—will be critical for biosensors development and will make sure that these tools can be deployed successfully in diverse environments.
Testing Concerns
Testing biosensors comes with its challenges, particularly due to variability in promoter responses under different environmental and lab conditions. To make sure that there is consistent performance, we need to validate our findings through repeated experiments across a range of different environmental variables. This will involve conducting titration assays, using control experiments, and employing cross-laboratory validation to identify any inconsistencies in promoter activity. We will also test the long-term stability of the biosensors and their effectiveness in real-world environments to confirm their reliability and robustness before any wider implementation action is taken. For instance, conducting experiments that are contained in bioreactor facilities and testing results in a human reflecting system before implementing these strategies in real world conditions. Additionally, it is important to consider the fact that the strain of E. coli which is being used isn’t pathogenic but it is important to be careful.
References
[1] “Carbaryl General Fact Sheet.” National Pesticide Information Center, http://npic.orst.edu/factsheets/carbarylgen.html. Accessed 12 September 2024;
[2] “PFOS (Perfluorooctane Sulfonate or Perfluorooctane Sulfonic Acid) - Proposition 65 Warnings Website.” P65Warnings.ca.gov, https://www.p65warnings.ca.gov/fact-sheets/pfos-perfluorooctane-sulfonate-or-perfluorooctane-sulfonic-acid. Accessed 15 September 2024;
[3] “Lovastatin.” MedlinePlus, https://medlineplus.gov/druginfo/meds/a688006.html. Accessed 12 September 2024;
[4] McDonald, Christopher. “Best Soil for Cannabis Seedlings: Sprouting Success.” Happy Hydro, 18 October 2023, https://www.happyhydro.com/blogs/growing-cannabis/best-soil-for-cannabis-seedlings. Accessed 12 September 2024.
