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    Bioinformatics, Computational and Systems Biology

    Finding Monoamine Interacting Proteins in Micrococcus luteus for Use in Biosensors

    (A-18) Finding Monoamine Interacting Proteins in Micrococcus luteus for Use in Biosensors

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row A - Poster # 18
    Introduction:: Serotonin, norepinephrine, and dopamine are monoamine neurotransmitters that play a pivotal role in regulating bodily functions: motor control, mental and gut health [1]. Their continuous monitoring fills a sizeable medical need, and the development of biosensors against these chemicals represents a milestone in personalized medicine. This development depends on the discovery of proteins, enzymes, or transcription factors (TFs), that respond to the presence of monoamines in a specific manner as we have recently developed the use of allosteric TFs as electronic sensors [2-4]. We present the framework for testing the tolerance of Micrococcus luteus against increasing concentrations of the aforementioned monoamines before cells exhibit a significant decrease in growth. Then, we extract samples to conduct RNA-seq analyses and identify which genes are differentially expressed in the presence of the monoamines to later express and purify the proteins, TFs, or enzymes coded by differentially expressed transcripts for their use in biosensors. We found the maximum tolerated concentration of the aforementioned monoamines for this strain. Moreover, we identified the three representative phases of growth and collected samples for RNA extraction and library preparation. Monoamine oxidases have previously been used to detect serotonin and norepinephrine but with relatively low specificity [5]. We anticipate the expansion of our enzyme library to incorporate ones that bind with higher specificity to tested monoamines, enabling the development of serotonin, norepinephrine, and dopamine biosensors for applications in mental and gut health and neurodegenerative disease diagnostics.

    Materials and Methods::

    M. luteus was streaked on an LB Agar plate and incubated at 30°C until single colonies were present on the plate (Fig. 1A). Single colonies were then used to inoculate starter cultures in Lactate Minimal Medium [6] until they were dense (OD600 > 1.00). Growth culture was inoculated with starter culture (1% inoculum) and exposed to increasing concentrations of monoamines in 96-well tissue culture plates (CELLTREAT) incubated at 30°C in a FluoSTAR Omega Microplate Reader for 48 hours to determine the concentration at which the growth would exhibit a significant decrease in optical density. Once the threshold concentrations of tested monoamines were identified, experiments were scaled up to 50.00 mL cultures and incubated at 30°C at 200 rpm. Samples for RNA processing were taken at lag, log, and stationary phases.



    Results, Conclusions, and Discussions::

    We observed a dose-dependent response by M. luteus to monoamines. M. luteus exhibited tolerance towards dopamine, norepinephrine, and serotonin at 2.65, 11.20, and 2.85 mM, respectively. Higher concentrations were likely toxic as they considerably affected the phenotype of M. luteus (Fig. 1B). In addition to optical density, we determined the specific growth rate as a guide to take the RNA-seq samples at growth rate thresholds for the three phases (Fig. 1C). We have extracted and purified RNA samples and are currently preparing library preparations for genome sequencing. After receiving our RNA-seq results, we expect to find several genes involved in the direct recognition of tested amines, some of which can be also related to the catabolism of monoamines in M. luteus as we have corroborated by our previous findings with Pseudomonas Aeruginosa (Fig. 1D). Proteins coded by some of these genes will be evaluated in terms of their feasibility to be incorporated into wearable biosensors for the detection and monitoring of monoamine levels in the human body. 


    We have outlined a pipeline for the screening, identification, and potential use of M. luteus proteins for later application in biosensors. In summary, the screening approach we have outlined lays the groundwork for identifying proteins that can later be used to develop wearable biosensors for use in personalized medicine and continuous monitoring.



    Acknowledgements (Optional): :

    References (Optional): : [1] Liu, T. J Art Int Med Sci. 2020, 1(1-2), 1-14. [2] Grazon, C. Nat Comm. 2020, 11(1), 1276. [3] Nguyen, Thuy T., Small 16.17. 2020, 1907522. [4] Parate, K, 2021. (unpublished data). [5] Chen, M. Adv Healthc Mat. 2020. 9(17), 2000403. [6] Kaprelyants A. J Appl Microbiol. 1992, 72, 410–422. [7] Tierrafria, V, 2022. (unpublished data)