<p>After separately verifying the effectiveness of the muscone molecular switch and the lactic acid secretion system in Saccharomyces cerevisiae, we constructed a complete therapeutic system within the yeast. We linked the lactate dehydrogenase behind the pFUS1 promoter, which is downstream of the mating pathway in Saccharomyces cerevisiae. For details, refer to design. The muscone receptor, the modified Gα protein, and the lactate dehydrogenase were simultaneously introduced into Saccharomyces cerevisiae. We tested the efficacy of the complete therapeutic system by using the same induction scheme as the muscone molecular switch and the WST colorimetric method to measure the supernatant content of D-lactic acid. For details, refer to protocol.</p>
<p>In the galactose-induced experimental group, the content of D-lactic acid in the supernatant of the muscone-induced group was significantly higher than that in the control group without muscone. However, in the glucose control group, there was no significant difference in the content of D-lactic acid in the supernatant between the muscone-induced group and the control group without muscone. This preliminarily verified that the complete therapeutic system constructed in Saccharomyces cerevisiae is effective. At the same time, we also found that, similar to the previous results of the lactic acid secretion system alone, the carbon source in the culture medium used during the culture and induction process has a significant impact on the lactic acid secretion results of Saccharomyces cerevisiae. Glucose promotes the background rate of lactic acid synthesis compared to galactose. In addition, we found that there were significant differences in lactic acid secretion values among different strains under the same induction conditions. We speculate that this may be related to the growth status of different strains and the copy number of the transformed plasmid.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 14 Muscone-induced lactate measurement results of the treatment system. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)</p>
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<p>Subsequently, we plan to replace the muscone receptor and the modified Gα protein expression promoter with a constitutive promoter, thereby eliminating the interference caused by differences in culture medium carbon source components during the experimental process. Later, we will transfer the related genes of the system into the genome of Saccharomyces cerevisiae to avoid the inter-group differences caused by plasmid copy number. At the same time, we will screen dominant strains for subsequent experiments to minimize the interference of strain differences on the results.</p>
