We have been working tirelessly for the most part of 2021 to make GutLux the best version it can be and we are proud of the progress we have made! As with any other student project, we also faced time constraints and a lack of access to some resources which would have enhanced our project to a higher level. In this wiki page we will talk about what can be done in the immediate and distant future to improve GutLux.
We set out to build a novel research tool that can detect tryptophan metabolites and produce fluorescence. To achieve this we opted to work with the AhR-ARNT transcription factor system. The system of choice is native to us humans and as such the proteins involved - Aryl hydrocarbon Receptor (AhR), Aryl hydrocarbon Receptor Nuclear Translocator (ARNT) and Aryl hydrocarbon Receptor Interacting Protein (AIP) - are difficult to express in our expression systems - Escherichia coli and Saccharomyces cerevisiae. We tried to work our way around by using truncated versions of AhR and ARNT for E. coli, whilst keeping the original coding sequences for S. cerevisiae. Though not ideal, it was necessary due to our short time frame. As such, we would like to continue optimizing and improving our expression systems.
It begins with successfully transforming our Level 2 plasmid constructs and our superfolder green fluorescent protein (sfGFP) reporter plasmid. If the transformations are successful, meaning desired AhR, ARNT and AIP are expressed, we would like to induce the expression of the sfGFP reporter gene by introducing different concentrations of metabolites into the microbial environment. We would then correlate the intensity of fluorescence to the concentration of metabolite added.
After our system is shown to function as designed, we would like to improve the specificity of AhR towards our target metabolites - kynurenic acid and tryptamine. We have identified the key amino acid residues of the binding site involved in the interaction between AhR and the metabolites through computational modeling, with the help of Rosetta and Autodock Vina. More details on our model can be found here. We would like to mutate these residues to see how they affect the binding of our target metabolite to AhR. This could also reveal if the system can accommodate a broader range of neuroactive molecules that naturally bind to AhR, like indole derivatives.
Our prototype design has laid the groundwork for the development of a functional, miniature-scale electronic system, which is able to convert measurement data to an electrical signal and send it outside of the body through wireless communication. To take this design even further, we would continue the design of a miniature-scale electronic system based on our current design and our research for component optimization. After successful fluorescence production from our biological detection mechanism, we would test the intensity measurement with our chosen light-dependent resistor (LDR) and confirm adequate sensitivity. We would then adapt the system to fit the requirements of our biological component, and develop the final design for our electronics. We would also conduct practical tests to enhance signal strength and reduce potential noise, optimizing the system for in-body communication. Simultaneously, we would continue the development of our receiver device and develop it into a watch-like, portable device that is easy to use and ensures proper data collection.
Once we have optimized our detection mechanism inside the capsule, we would also strive to improve the overall GutLux capsule itself. We are currently looking for a semipermeable membrane that best allows for the flow of our metabolites into the capsule, and prevents the leakage of genetically modified organisms (GMOs) from our capsule. We would also like to provide a coating over our capsule to prevent premature detection of our metabolites before the capsule reaches the gut, and to also protect the membrane and our engineered cells from harsh stomach acids.
We see GutLux as a research tool, able to provide scientists with studies of overall trends of mental-health associated gut metabolites in large populations. Thus, we hope that one day, GutLux will participate in clinical studies and assist in creating an open-access information database of gut microbiota-associated metabolite concentrations to expand knowledge and shed light on this fascinating pathway.