Future Works

During each cycle of our project, the team has worked to optimize our devices in order to fulfill our goal in detecting and biodegrading 1,3,5-trinitro-1,3,5-triazinane (RDX) found in the Anones Lagoon in Vieques. We have come a long way since this project started and have made many advances. However, there are still some aspects that must be improved in order to reach our objective.

Among them, one of our ongoing efforts is to search for an operational promoter that is inducible by RDX. Our Device 1 previously contained promotor AlgD, induced by the presence of RDX, but not all of its homologous regulators were found in E. coli. Therefore, it was decided that for laboratory purposes a supplementary Device 1, containing an IPTG inducible promoter known as pLac-LuxI, would have to be created in order to execute the proof of concept of our Device 2. However, a design modification was generated for the Device 1 that will form part of our official circuit. In an experiment conducted by Lifshitz et. al, this group of scientists were able to design, construct, and test E.coli-based bioluminescent bioreporters for the detection of RDX. They were able to achieve this by performing a fusion between the promoters of the hmp (nitric oxide dioxygenase) and the hcp (a high-affinity nitric oxide reductase) E. coli gene, to the microbial bioluminescence luxCDABEG gene cassette. Their results suggest that each of the promoters had the ability to detect RDX, but a tandem that contained both showed an increased amount of transcription (Lifshitz et al., 2021). Therefore, the combination of both would be more optimal for the objective of our device. We plan on searching the right sequences of the hcp and hmp gene promoters in order to replace the algD promoter found in Device 1. We also aspire to become familiarized with the protocols needed to conduct double promoter expression and be able to fuse both promoters in order to have an effective detection of RDX.

Another improvement that we plan on pursuing is the implementation of an aptamer-based riboswitch for our device. Riboswitches are non-coding mRNA that are capable of both recognizing a specific small molecule compound and regulating the transcription and translation of downstream genes (Garst et al., 2011). According to Sawyze et al., riboswitches consist of two regions, an aptameric region responsible for binding the metabolite and the other region responsible for genetic regulation and expression. Some examples of riboswitch ligands are glycine, thiamine, flavin mononucleotides, S-adenosylmethionine, and guanine (2007). In our project, the RDX will act as a ligand. Therefore, when there is no RDX bound to the aptamer binding region, a three-dimensional conformation prevents access of the ribosome to the RBS. With this addition, we wish to have better control over the gene expression of our device. However, creating a riboswitch poses its own challenges. We plan to continue researching this topic and its viability for use in our genetic circuit.

Eventually, once we have been able to design and test our prototype, we would also like to perform a fluorometric assay in a 96-well plate reader and high-performance liquid chromatography (HPLC) to test the performance of the assembly of Device 1 with Device 2. We also wish to construct a graph that expresses the relationship between the concentration of RDX and the time to show our data. This will help us visualize the decline in concentration over time, allowing us to analyze its potential rate. The details of this plan are redacted in the Proof Concept.