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Overview


The MusCure project is a treatment scheme designed for inflammatory bowel disease (IBD), an intestinal autoimmune disease, using engineered bacteria for auxiliary therapy. We have designed a muscone gas molecule switch in Saccharomyces cerevisiae, which allows for the control of engineered bacteria to secrete lactic acid and inhibit the abnormal activation of immune cells, achieving the purpose of auxiliary treatment and greatly improving the treatment experience for patients. Compared to traditional treatment methods, MusCure has a lower treatment cost, minimal discomfort for patients during treatment, and more convenient treatment tools.

To verify the feasibility and social value of the MusCure project, as well as its potential future applications, we have evaluated and validated our project from three perspectives: 1. Previous literature research, 2.Experimental results, 3. Communication with relevant social stakeholders. Although we have put in a lot of effort in the project design and experimental process, due to the limitations of the time period and technical means, there are still many deficiencies in our project design and experimental results. In response to this, we have proposed some future solutions and methods.

Therapy system


For the therapy system, we have conducted verification of the principles and results in three parts: muscone molecular switches, lactate secretion, and the complete system.

Proof of Mucsone molecular switch

Previous literature research

We hope to improve the treatment experience for IBD patients, so we have chosen gas signals as the therapeutic switch for the engineered bacterial secretion system. Drawing inspiration from the research of the Ye Haifeng team[1], we selected the muscone molecular receptor they developed and designed it to be transferred into Saccharomyces cerevisiae, to create an engineered Saccharomyces cerevisiae muscone molecular switch.

The muscone receptor is a G protein-coupled receptor derived from mouse olfactory epithelial cells, and we need to integrate the muscone receptor with the existing G protein signaling pathway in Saccharomyces cerevisiae. Referencing the research of the Benjamin M Scott team[2], we have modified the signaling pathways in Saccharomyces cerevisiae according to their operational methods and achieved the creation of a muscone molecular switch in this model organism.

For more information, please refer to Description.

Experimental results

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fig 1 Muscone molecular switch fluorescence signal test

A. Galactose-induced, add muscone organic solution.

B. Galactose-induced, without muscone.

We have tested the functionality of the designed muscone molecular switch in Saccharomyces cerevisiae using the GFP reporter gene intensity. Additionally, we have reduced background noise from wild-type strains by knocking out the original mating pathway receptors in Saccharomyces cerevisiae.

For more information, please refer to Therapy system.

Communication with relevant social stakeholders

To assess the potential for further applications of the muscone molecular switch in Saccharomyces cerevisiae, we communicated with Bluepha Company, which specializes in developing new microbial fermentation materials through synthetic biology. Bluepha Company's feedback indicated that the muscone molecular switch in our modified Saccharomyces cerevisiae could serve as an alternative to the traditional methanol promoter used in Pichia pastoris fermentation systems. Muscone offers cost-effectiveness and higher safety compared to methanol, making it an attractive option for the design of innovative fermentation processes in Saccharomyces cerevisiae.

Shortcoming: Background signal noise still exists; There are significant differences in baseline expression between different strains.

Solution: Take further measures to modify the yeast genome to eliminate background noise; Introduce the musk ketone molecular switch gene into the yeast genome to avoid the impact of plasmid cloning variations, and screen for superior yeast strains for cultivation.

Proof of secretion of lactate

Previous literature research

We selected a small molecule drug that is relatively easy to biosynthesize and can be readily secreted to treat diseases by passing through intestinal wall cells. Through literature search, we found that Liliana M Sanmarco et. discovered a signaling pathway in which lactic acid inhibits the abnormal activation of immune cells[3]. In their study, they designed an engineered E. coli bacteria to secrete lactic acid and successfully suppressed dendritic cells and T cells in the intestinal tract. This provided a reference for our choice. We decided to introduce lactate dehydrogenase into brewing yeast, altering the anaerobic metabolism pathway to synthesize D-lactic acid for therapeutic purposes.

For more information, please refer to Description.

Experimental results

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fig 2 Galactose-induced D-lactic acid secretion system

We introduced a lactate dehydrogenase gene expressed by the galactose promoter into Saccharomyces cerevisiae and measured the content of D-lactic acid in the supernatant after induction. This validated that by introducing lactate dehydrogenase, we can alter the anaerobic metabolic pathway of S. cerevisiae to synthesize D-lactic acid, and the produced D-lactic acid can be secreted into the surrounding environment of the yeast.

For more information, please refer to Description.

Shortcoming: Experimental findings have shown that the nutritional composition of the yeast culture environment has a significant impact on the secretion of lactic acid; In the anaerobic environment, yeast can still produce alcohol, which may have an impact on the health of patients.

Solution:By further modifying the yeast's anaerobic metabolic pathways through genomic engineering, the impact of the nutritional composition of the culture environment on lactic acid secretion can be reduced; knockout of the alcohol dehydrogenase gene in the yeast genome.

Proof of whole system

Experimental results

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fig 3 Muscone-induced lactate measurement results of the treatment system. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)

We have simultaneously introduced the muscone molecular switch and the downstream lactate dehydrogenase into Saccharomyces cerevisiae, constructing a complete therapeutic system within the organism. After induction with muscone, we tested the content of D-lactic acid in the culture supernatant. The experimental results demonstrate that our complete therapeutic system can achieve specific secretion of lactic acid. For further discussion on the experimental results, please refer to Therapy system

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