Implementation

Cost

The cost of the promoter collection was $2,535.00 [3], and cultivating E. coli is fairly straightforward. The price of the molecules tested for the biosensor are as follows:

All molecules were purchased through Fisher Scientific [1], with the exception of CND, which was purchased through Santa Cruz Biotechnology [2]

Challenges

Our team evaluated many possible routes for developing a biosensor that would be applicable in so many fields. We specifically wanted to develop this biosensor to be as versatile as possible but we found that many attempts to actually degrade these molecules usually fell short of this goal, so instead we aimed to act as the foundational first step in detecting these molecules in order to help future scientists on this path.

1. Initial Screening

When measuring fluorescent values of molecule-promoter pairs, lack of E. Coli growth produced some wells with an OD600 fluorescent value less than 0.1. For cleaner data, we disregarded wells with a sfGFP/OD600 value less than 0.1 and chose an original cutoff of sfGFP/OD600 < 1.5. However, because of the lack of sufficient molecule-promoter pairs that produced a significant fluorescent value, the sfGFP/OD600 cutoff was lowered to 1.35.

2. Titrations

An increasing fluorescence proportional to increasing molecular concentrations was expected, but following titrations, this trend was not observed. During the initial screening process, however, these molecule-promoter pairs did produce a high fluorescence. When these pairs were tested with the same molecular concentrations in titrations, the sfGFP/OD600 values were significantly lower than that of the initial screening process.

Initially, we noticed that the cell density of the E. Coli strains were not significant enough and produced an OD600 value less than 0.1, which meant that the cell density was not enough to produce a significant fluorescent response. To combat this, we regrew the strains and measured the density to ensure sufficient growth.

Once the titrations were rerun, we did notice a minor improvement in results, but there was still a linear trend in fluorescent response rather than the expected exponential trend. We found that this was because the LB broth used to grow the E. coli strains was contaminated. Using a sterilized, autoclaved, and fresh media, we reran the titration.

The new titration values were slightly higher than the previous run, but they were still significantly smaller than the initial screening values. We noticed that some of the E. coli strains used were several days old in titration, but fresh in initial screening. We then reran the titration with newly regrown strains. While this did produce an even higher titration value than before, it still did not match the initial screening values.

We decided to rerun the initial screening to ensure no problems arose in the original process. We also tested the fluorescence levels of a single promoter with each of the 10 molecules, with the expectations that multiple molecules cannot induce the same promoter. Some promoters did indeed only produce one significant fluorescent response when tested with all the molecules. However, other promoters indicated a high fluorescent response for most molecules, indicating their fluorescence wasn’t in response to purely a particular molecule and may be induced by other factors.

We also found that some fluorescent plates were not matched to their promoters, explaining the initial lack of increasing fluorescence with increasing molecule concentration and lower levels of fluorescence compared to initial screening. Fixing these incorrect matchings, we reran the titration and observed an improved response. We could then further our experiment by selecting specific molecule-promoter combinations with the best fluorescence trend produced by the titrations.

End Users

The end users for our project are researchers who can use our biomarker for potential further research. As a foundational first step, our biosensor will hopefully help researchers identify harmful environmental molecules that can aid them in the field, and can later be used to research potential options for degrading these molecules. Being able to detect the presence of these molecules in the environment is the first step in making progress toward proper handling of these toxic chemicals in the environment, which can damage animals and ecosystems. If our project is successful, we can not only help scientists implement this novel approach in their work but will also contribute valuable research to the science community to protect the Earth’s environment and other processes by addressing molecules such as propoxur, an insecticide, and Perfluorooctane sulfonate, a man made chemical found in PFAS. We acknowledge that our team has not found a perfect solution to remove all of these harmful chemicals from the environment completely, but we hope that detection will serve as a valuable first step to help other researchers working with these molecules to increase efficiency as well as help them degrade these molecules in the future. We hope that through this novel approach, we will hopefully have the potential to develop a key tool for researchers in the future, and we will keep developing more accurate biosensors for the detection of these molecules to allow for more effective monitoring in the future.

References

[1] Laboratory and Production Essentials | Fisher Scientific. www.fishersci.com/us/en/home.html.

[2] Antibodies, Gene Editors, Chemicals and Lab Supplies for Research. www.scbt.com/home.

[3] E. Coli Promoter Collection. horizondiscovery.com/en/non-mammalian-research-tools/products/e-coli-promoter-collection.