<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 1 The design of Muscone-gated molecular switch in Saccharomyces cerevisiae</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 1 The design of Muscone-gated molecular switch in <i>Saccharomyces cerevisiae</i></p>
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<p>One of our standout contributions to synthetic biology tools is the construction of a Muscone-gated molecular switch in Saccharomyces cerevisiae BBa_K5187006. The muscone receptor is a type of GPCR derived from mice. We designed the corresponding Gα protein by modifying the amino acid sequence so that it can trigger the mating pathway in Saccharomyces cerevisiae, thereby further inducing the expression of downstream genes.</p>
<p>It is worth noting that this molecular switch in Saccharomyces cerevisiae is an original contribution from our team. Although in our experiment it was applied to secrete lactic acid for the treatment of IBD, this downstream element can be replaced with any other gene to achieve different biological functions. We believe that this minute, efficient, and cost-effective molecular switch will have broad application scenarios in related industries.</p>
<p>One of our standout contributions to synthetic biology tools is the construction of a Muscone-gated molecular switch in <i>Saccharomyces cerevisiae</i> BBa_K5187006. The muscone receptor is a type of GPCR derived from mice. We designed the corresponding Gα protein by modifying the amino acid sequence so that it can trigger the mating pathway in <i>Saccharomyces cerevisiae</i>, thereby further inducing the expression of downstream genes.</p>
<p>It is worth noting that this molecular switch in <i>Saccharomyces cerevisiae</i> is an original contribution from our team. Although in our experiment it was applied to secrete lactic acid for the treatment of IBD, this downstream element can be replaced with any other gene to achieve different biological functions. We believe that this minute, efficient, and cost-effective molecular switch will have broad application scenarios in related industries.</p>
<p>Simultaneously, we have conducted molecular dynamics simulations to predict the binding of muscone molecules to their receptors, providing a more detailed and quantitative explanation of the biological process of the muscone-gated molecular switch. This aims to assist future research teams in obtaining more molecular dynamics information about this molecular switch and developing more functions of the switch. For more information, please refer to <ahref="https://2024.igem.wiki/Tsinghua/model#topic2"style="color: #FF5151">Model: Binding</a>.</p>
<p>For more information about the Muscone-gated molecular switch in Saccharomyces cerevisiae, please refer to <ahref="https://2024.igem.wiki/Tsinghua/parts"style="color: #FF5151">Parts</a>.</p>
<p>For more information about the Muscone-gated molecular switch in <i>Saccharomyces cerevisiae</i>, please refer to <ahref="https://2024.igem.wiki/Tsinghua/parts"style="color: #FF5151">Parts</a>.</p>
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<p>In order to popularize knowledge about molecular biology experiments used in synthetic biology, we have registered accounts on various video media platforms such as Tiktok, Bilibili, and YouTube. Based on the experimental content of our project, we have produced and released a series of instructional videos on molecular biology experimental skills in both Chinese and English versions, helping more people learn related experimental techniques.</p>
<p>For more information about the instructional videos, please refer to <ahref="https://2024.igem.wiki/Tsinghua/education#Public"style="color: #FF5151">Education: Public</a>.</p>
<h3>About new experimental protocol</h3>
<p>Our project uses Saccharomyces cerevisiae as the chassis organism. During the design and experimental process of our project, we have optimized and improved the original experimental operation procedures for the transformation, culture, and induction of Saccharomyces cerevisiae. This not only helped us achieve better results in the project experiments but can also save experimental time and costs for future iGEM teams and members who wish to use brewing yeast as the chassis organism.</p>
<p>Our project uses <i>Saccharomyces cerevisiae</i> as the chassis organism. During the design and experimental process of our project, we have optimized and improved the original experimental operation procedures for the transformation, culture, and induction of <i>Saccharomyces cerevisiae</i>. This not only helped us achieve better results in the project experiments but can also save experimental time and costs for future iGEM teams and members who wish to use brewing yeast as the chassis organism.</p>
<p>For more information about the new protocol, please refer to <ahref="https://2024.igem.wiki/Tsinghua/therapy-system#Protocol"style="color: #FF5151">Protocol</a>.</p>
<p>According to our design, therapeutic engineered yeast will be produced in a large-scale specialized microbiological facility. Specialized facilities must have the appropriate equipment and trained personnel. What’s more, the laboratory and premises during the production process must adhere to international biosafety standards and ensure strict biosafety levels, such as BSL-2 or BSL-3. The cultivation of Saccharomyces cerevisiae must take place in controlled bioreactors to minimize the risk of leakage. Personnel must undergo specialized training and strictly follow operating procedures, including wearing protective gear and using biosafety cabinets. Waste must be handled and disposed of in accordance with the standards for biohazardous waste to prevent any leakage of biological materials.</p>
<p>According to our design, therapeutic engineered yeast will be produced in a large-scale specialized microbiological facility. Specialized facilities must have the appropriate equipment and trained personnel. What’s more, the laboratory and premises during the production process must adhere to international biosafety standards and ensure strict biosafety levels, such as BSL-2 or BSL-3. The cultivation of <i>Saccharomyces cerevisiae</i> must take place in controlled bioreactors to minimize the risk of leakage. Personnel must undergo specialized training and strictly follow operating procedures, including wearing protective gear and using biosafety cabinets. Waste must be handled and disposed of in accordance with the standards for biohazardous waste to prevent any leakage of biological materials.</p>
<p>We introduced muscone gas molecule receptors derived from mouse olfactory epithelial cells into chassis bioengineered bacteria. The muscone gas molecule receptor is a G protein coupled receptor in eukaryotic cells, and we chose Saccharomyces cerevisiaet as the chassis bioengineering bacterium. By modifying the mating pathway of Saccharomyces cerevisiae, the muscone gas molecule receptor is integrated into the signaling pathway of Saccharomyces cerevisiae. And downstream of the modified mating pathway, lactate dehydrogenase was introduced to alter the anaerobic metabolism pathway of Saccharomyces cerevisiae, synthesizing lactate for the treatment of IBD disease.</p>
<p>Our experimental design consists of two parts: the verification of the mating pathway in Saccharomyces cerevisiae containing muscone gas molecule switches and the modification of the Saccharomyces cerevisiae genome. We independently verified each component of the mating pathway in the modified Saccharomyces cerevisiae and ultimately integrated them. Additionally, we knocked out the original receptor of the Saccharomyces cerevisiae mating signal to eliminate signal interference caused by the yeast’s own growth.</p>
<p>We introduced muscone gas molecule receptors derived from mouse olfactory epithelial cells into chassis bioengineered bacteria. The muscone gas molecule receptor is a G protein coupled receptor in eukaryotic cells, and we chose <i>Saccharomyces cerevisiae</i> as the chassis bioengineering bacterium. By modifying the mating pathway of <i>Saccharomyces cerevisiae</i>, the muscone gas molecule receptor is integrated into the signaling pathway of <i>Saccharomyces cerevisiae</i>. And downstream of the modified mating pathway, lactate dehydrogenase was introduced to alter the anaerobic metabolism pathway of <i>Saccharomyces cerevisiae</i>, synthesizing lactate for the treatment of IBD disease.</p>
<p>Our experimental design consists of two parts: the verification of the mating pathway in <i>Saccharomyces cerevisiae</i> containing muscone gas molecule switches and the modification of the <i>Saccharomyces cerevisiae<i> genome. We independently verified each component of the mating pathway in the modified <i>Saccharomyces cerevisiae</i> and ultimately integrated them. Additionally, we knocked out the original receptor of the <i>Saccharomyces cerevisiae</i> mating signal to eliminate signal interference caused by the yeast’s own growth.</p>
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<h3>Therapy system</h3>
<p>Our designed Therapy system primarily consists of three parts: </p>
<p>We selected muscone gas molecules as the upstream control signal for our therapy system. We used the muscone receptor sequence from mouse olfactory epithelial cells, as employed by the Ye Haifeng team <sup>[1]</sup>; for details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank"style="color: #FF5151 ;">description</a>, and introduced it into the plasmid system expressed in Saccharomyces cerevisiae. We chose the mating pathway in Saccharomyces cerevisiae as the transmission pathway for the muscone signal within Saccharomyces cerevisiae. Based on the Benjamin M Scott team's optimization<sup>[2]</sup>, we replaced the C-terminal five amino acids of the Gα protein in the original mating pathway, allowing the muscone receptor to be integrated into the Saccharomyces cerevisiae mating pathway.</p>
<p>We used the galactose promoter to induce the expression of the muscone signal receptor and the optimized Gα protein, and screened the successfully transformed Saccharomyces cerevisiae with a His nutritional deficiency. By controlling the induction conditions of galactose and muscone, we tested the effectiveness of the muscone gas molecule switch. For details, please refer to the protocol.</p>
<p>We selected muscone gas molecules as the upstream control signal for our therapy system. We used the muscone receptor sequence from mouse olfactory epithelial cells, as employed by the Ye Haifeng team <sup>[1]</sup>; for details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank"style="color: #FF5151 ;">description</a>, and introduced it into the plasmid system expressed in <i>Saccharomyces cerevisiae</i>. We chose the mating pathway in <i>Saccharomyces cerevisiae</i> as the transmission pathway for the muscone signal within <i>Saccharomyces cerevisiae</i>. Based on the Benjamin M Scott team's optimization<sup>[2]</sup>, we replaced the C-terminal five amino acids of the Gα protein in the original mating pathway, allowing the muscone receptor to be integrated into the <i>Saccharomyces cerevisiae</i> mating pathway.</p>
<p>We used the galactose promoter to induce the expression of the muscone signal receptor and the optimized Gα protein, and screened the successfully transformed <i>Saccharomyces cerevisiae</i> with a His nutritional deficiency. By controlling the induction conditions of galactose and muscone, we tested the effectiveness of the muscone gas molecule switch. For details, please refer to the protocol.</p>
<p>Aim:</p>
<p>To validate the effectiveness of the muscone gas molecule switch in Saccharomyces cerevisiae.</p>
<p>To validate the effectiveness of the muscone gas molecule switch in <i>Saccharomyces cerevisiae</i>.</p>
<p>We chose the mating pathway in Saccharomyces cerevisiae as the conduit for muscone signaling in yeast. Using the mating pathway’s pFUS1 promoter, we expressed the downstream lactate dehydrogenase to alter the anaerobic metabolic pathway of Saccharomyces cerevisiae, secreting lactic acid for the treatment of IBD<sup>[3]</sup>. Initially, we designed a plasmid with the pFUS1 promoter expressing the GFP reporter gene and screened the successfully transformed yeast using Ura nutritional deficiency. We then tested the effectiveness of the muscone molecular switch using confocal microscopy; for details, please refer to the protocol. Subsequently, we designed the pFUS1 promoter to express lactate dehydrogenase from E. coli. By co-transforming it with Muscone Receptor & Gα (pESC) into Saccharomyces cerevisiae, we achieved the construction of the complete pathway.</p>
<p>We chose the mating pathway in <i>Saccharomyces cerevisiae</i> as the conduit for muscone signaling in yeast. Using the mating pathway’s pFUS1 promoter, we expressed the downstream lactate dehydrogenase to alter the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i>, secreting lactic acid for the treatment of IBD<sup>[3]</sup>. Initially, we designed a plasmid with the pFUS1 promoter expressing the GFP reporter gene and screened the successfully transformed yeast using Ura nutritional deficiency. We then tested the effectiveness of the muscone molecular switch using confocal microscopy; for details, please refer to the protocol. Subsequently, we designed the pFUS1 promoter to express lactate dehydrogenase from E. coli. By co-transforming it with Muscone Receptor & Gα (pESC) into <i>Saccharomyces cerevisiae</i>, we achieved the construction of the complete pathway.</p>
<p>Aim:</p>
<p>To check the reporter signals downstream of the muscone molecular switch.</p>
<p>To check the synthesis of the secretion system downstream of the muscone molecular switch.</p>
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<h3>Lactate secretion</h3>
<p>Lactic acid is the small molecule we selected to target the abnormally activated autoimmune cells in IBD diseases. For details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank">description</a>. We chose lactate dehydrogenase from E. coli to alter the anaerobic metabolic pathway of Saccharomyces cerevisiae to synthesize and secrete D-lactic acid. We used the galactose promoter to induce the expression of lactate dehydrogenase and screened the successfully transformed yeast with a Ura nutritional deficiency. By controlling the induction with galactose and glucose and establishing gradients of induction time and post-induction culture time, we tested the synthesis and secretion of lactic acid and searched for the optimal induction conditions for lactic acid secretion. For details, please refer to the protocol.</p>
<p>Lactic acid is the small molecule we selected to target the abnormally activated autoimmune cells in IBD diseases. For details, please refer to the <ahref="https://2024.igem.wiki/tsinghua/description"target="_blank">description</a>. We chose lactate dehydrogenase from E. coli to alter the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i> to synthesize and secrete D-lactic acid. We used the galactose promoter to induce the expression of lactate dehydrogenase and screened the successfully transformed yeast with a Ura nutritional deficiency. By controlling the induction with galactose and glucose and establishing gradients of induction time and post-induction culture time, we tested the synthesis and secretion of lactic acid and searched for the optimal induction conditions for lactic acid secretion. For details, please refer to the protocol.</p>
<p>Aim:</p>
<p>To test the effectiveness of the lactate secretion system in Saccharomyces cerevisiae.</p>
<p>To test the effectiveness of the lactate secretion system in <i>Saccharomyces cerevisiae</i>.</p>
<p>To explore the optimal induction conditions for the lactate secretion system.</p>
<p>To avoid interference from the mating signals of Saccharomyces cerevisiae's own growth on the signal transduction controlled by the muscone molecule and to ensure biological safety, we have made modifications to the genome of Saccharomyces cerevisiae. This is reflected in our knockout of the original receptor STE2 in the mating pathway of Saccharomyces cerevisiae. Our knockout system includes a gRNA targeting the STE2 gene and the Cas9 protein.</p>
<p>To avoid interference from the mating signals of <i>Saccharomyces cerevisiae</i>'s own growth on the signal transduction controlled by the muscone molecule and to ensure biological safety, we have made modifications to the genome of <i>Saccharomyces cerevisiae</i>. This is reflected in our knockout of the original receptor STE2 in the mating pathway of <i>Saccharomyces cerevisiae</i>. Our knockout system includes a gRNA targeting the STE2 gene and the Cas9 protein.</p>
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<h3>Origin receptor knock-out</h3>
<p>We expressed the gRNA targeting the STE2 receptor gene and the Cas9 protein through a constitutive promoter and screened the successfully transformed yeast using leu nutritional deficiency. The genome of the successfully transformed Saccharomyces cerevisiae strains was sequenced to screen for strains with a successful knockout of STE2, followed by the transformation of the muscone molecular switch signaling pathway.</p>
<p>We expressed the gRNA targeting the STE2 receptor gene and the Cas9 protein through a constitutive promoter and screened the successfully transformed yeast using leu nutritional deficiency. The genome of the successfully transformed <i>Saccharomyces cerevisiae</i> strains was sequenced to screen for strains with a successful knockout of STE2, followed by the transformation of the muscone molecular switch signaling pathway.</p>
<p>Aim:</p>
<p>To remove the interference of Saccharomyces cerevisiae's own growth and mating signals on the secretory system.</p>
<p>To remove the interference of <i>Saccharomyces cerevisiae</i>'s own growth and mating signals on the secretory system.</p>
<p>In our system, the muscone molecule serves as the signal controlling the secretion system. Introducing the muscone molecular signal switch into the signaling pathway of Saccharomyces cerevisiae is one of our core tasks. You can view our experimental design through the design.</p>
<p>In our system, the muscone molecule serves as the signal controlling the secretion system. Introducing the muscone molecular signal switch into the signaling pathway of <i>Saccharomyces cerevisiae</i> is one of our core tasks. You can view our experimental design through the design.</p>
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<h3>Muscone molecular switch</h3>
<p>To verify the effectiveness of the muscone molecular switch in Saccharomyces cerevisiae, we used the GFP reporter gene to reflect the signal intensity downstream of the muscone molecular switch. By using the galactose promoter to induce the expression of the muscone molecular switch, we established a glucose-induced control group during the induction process. Additionally, we set up control groups with and without muscone under two different carbon source induction conditions. Details of the induction experiment can be found in the protocol. We captured fluorescence signal images of different groups of Saccharomyces cerevisiae under a confocal microscope and conducted quantitative analysis of relative fluorescence intensity and fluorescence proportion.</p>
<p>In the galactose-induced experimental group, the fluorescence intensity and proportion of the GFP reporter gene under muscone induction were significantly higher than those in the control group without muscone. In the glucose control group, there was no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone-induced experimental group and the control group. The experiment preliminarily proves the effectiveness of the introduced muscone molecular switch in Saccharomyces cerevisiae.</p>
<p>At the same time, we found that compared to the galactose-induced experimental group, the glucose control group showed a higher background noise of mating signals in Saccharomyces cerevisiae. In our subsequent experimental design, we knocked out the original receptor of the Saccharomyces cerevisiae mating signal pathway, which reduced the background signal intensity of the Saccharomyces cerevisiae mating signal pathway and improved the reliability of the system.</p>
<p>To verify the effectiveness of the muscone molecular switch in <i>Saccharomyces cerevisiae</i>, we used the GFP reporter gene to reflect the signal intensity downstream of the muscone molecular switch. By using the galactose promoter to induce the expression of the muscone molecular switch, we established a glucose-induced control group during the induction process. Additionally, we set up control groups with and without muscone under two different carbon source induction conditions. Details of the induction experiment can be found in the protocol. We captured fluorescence signal images of different groups of <i>Saccharomyces cerevisiae</i> under a confocal microscope and conducted quantitative analysis of relative fluorescence intensity and fluorescence proportion.</p>
<p>In the galactose-induced experimental group, the fluorescence intensity and proportion of the GFP reporter gene under muscone induction were significantly higher than those in the control group without muscone. In the glucose control group, there was no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone-induced experimental group and the control group. The experiment preliminarily proves the effectiveness of the introduced muscone molecular switch in <i>Saccharomyces cerevisiae</i>.</p>
<p>At the same time, we found that compared to the galactose-induced experimental group, the glucose control group showed a higher background noise of mating signals in <i>Saccharomyces cerevisiae</i>. In our subsequent experimental design, we knocked out the original receptor of the <i>Saccharomyces cerevisiae</i> mating signal pathway, which reduced the background signal intensity of the <i>Saccharomyces cerevisiae</i> mating signal pathway and improved the reliability of the system.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 8 Muscone molecular switch fluorescence signal test, A. Galactose-induced, add muscone organic solution. B. Galactose-induced, without muscone. C. Glucose control group, add muscone organic solution. D. Glucose control group, without muscone.</p>
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<h3>Lactate secretion</h3>
<p>In our system, the lactic acid molecule is the small molecule that ultimately treats IBD diseases and alleviates the abnormal activation of autoimmune cells. We have altered the anaerobic metabolic pathway of Saccharomyces cerevisiae by inducing the expression of lactate dehydrogenase from E. coli with galactose, and tested the effectiveness of the secretion system. You can view our experimental design through the design.</p>
<p>We used the galactose promoter to control the expression of lactate dehydrogenase within Saccharomyces cerevisiae. We established a glucose-induced control group during the induction process. And we also conducted an induction experiment with untransformed wild-type yeast as a control to exclude the background signal noise of Saccharomyces cerevisiae. Details of the induction experiment can be found in the protocol. After induction, the bacterial solution was centrifuged, and the supernatant was collected. The WST colorimetric method was used to measure the lactic acid content in the supernatant of different groups. The absorbance of the colorimetric reaction was recorded at 455 nm, which reflects the concentration of D-lactic acid in the supernatant.</p>
<p>The experimental results preliminarily demonstrate that in the transformed groups, galactose induction led to the expression of lactate dehydrogenase, which successfully altered the anaerobic metabolic pathway of Saccharomyces cerevisiae to synthesize D-lactic acid. Meanwhile, after the synthesis of D-lactic acid, it can be secreted into the surrounding environment of the Saccharomyces cerevisiae.</p>
<p>In our system, the lactic acid molecule is the small molecule that ultimately treats IBD diseases and alleviates the abnormal activation of autoimmune cells. We have altered the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i> by inducing the expression of lactate dehydrogenase from E. coli with galactose, and tested the effectiveness of the secretion system. You can view our experimental design through the design.</p>
<p>We used the galactose promoter to control the expression of lactate dehydrogenase within <i>Saccharomyces cerevisiae</i>. We established a glucose-induced control group during the induction process. And we also conducted an induction experiment with untransformed wild-type yeast as a control to exclude the background signal noise of <i>Saccharomyces cerevisiae</i>. Details of the induction experiment can be found in the protocol. After induction, the bacterial solution was centrifuged, and the supernatant was collected. The WST colorimetric method was used to measure the lactic acid content in the supernatant of different groups. The absorbance of the colorimetric reaction was recorded at 455 nm, which reflects the concentration of D-lactic acid in the supernatant.</p>
<p>The experimental results preliminarily demonstrate that in the transformed groups, galactose induction led to the expression of lactate dehydrogenase, which successfully altered the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i> to synthesize D-lactic acid. Meanwhile, after the synthesis of D-lactic acid, it can be secreted into the surrounding environment of the <i>Saccharomyces cerevisiae</i>.</p>
<p>Taking into account that different carbon sources for induction and cultivation systems may affect the growth of Saccharomyces cerevisiae and the working rate of lactate dehydrogenase. We further established different combinations of induction and cultivation with glucose and galactose groups based on previous experiments, and set up a time gradient to sample the supernatant of Saccharomyces cerevisiae cultures for the measurement of D-lactic acid concentration.</p>
<p>The experimental results show that, compared to the galactose group, the glucose group has a non-specific promotional effect on the secretion of D-lactic acid by Saccharomyces cerevisiae. In the short term of induction and cultivation, glucose will synthesize more D-lactic acid by promoting yeast growth more strongly and accelerating the reaction rate of lactate dehydrogenase. However, in longer-term induction experiments, the galactose group will ultimately synthesize more D-lactic acid due to the specific induction of lactate dehydrogenase expression. This experimental result confirms the impact of different carbon source cultivation systems on the secretion system. To eliminate unnecessary influences as much as possible, we adopted a muscone molecular switch expressed by a constitutive promoter in subsequent experiments, removing the galactose-induced experimental step.</p>
<p>Taking into account that different carbon sources for induction and cultivation systems may affect the growth of Saccharomyces cerevisiae and the working rate of lactate dehydrogenase. We further established different combinations of induction and cultivation with glucose and galactose groups based on previous experiments, and set up a time gradient to sample the supernatant of <i>Saccharomyces cerevisiae</i> cultures for the measurement of D-lactic acid concentration.</p>
<p>The experimental results show that, compared to the galactose group, the glucose group has a non-specific promotional effect on the secretion of D-lactic acid by <i>Saccharomyces cerevisiae</i>. In the short term of induction and cultivation, glucose will synthesize more D-lactic acid by promoting yeast growth more strongly and accelerating the reaction rate of lactate dehydrogenase. However, in longer-term induction experiments, the galactose group will ultimately synthesize more D-lactic acid due to the specific induction of lactate dehydrogenase expression. This experimental result confirms the impact of different carbon source cultivation systems on the secretion system. To eliminate unnecessary influences as much as possible, we adopted a muscone molecular switch expressed by a constitutive promoter in subsequent experiments, removing the galactose-induced experimental step.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 11 Changes in lactic acid secretion with different carbon source combinations in short-term culture.</p>
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<h3>Origin receptor knock-out</h3>
<p>Based on the previous experimental results of the muscone molecular switch signal, we designed a knockout system targeting the original receptor STE2 of the mating pathway in Saccharomyces cerevisiae, including the gRNA and Cas9 protein targeting STE2. We performed sequencing on the successfully transformed strains and confirmed that base deletions and frameshift mutations occurred in the original receptor STE2 sequence. We retransformed the strains with successful STE2 knockout with MOR215&Ga-pESC and pFUS1 promoter-GFP-pYES and repeated the previous induction experimental protocol. The results of the muscone analysis switch signal intensity in the knockout Saccharomyces cerevisiae strain were obtained under a confocal microscope.</p>
<p>The experimental results show that under the muscone induction condition in the galactose group, the fluorescence intensity and proportion of the GFP reporter gene remained significantly higher than that of the control group without muscone. In the glucose group, there was still no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone induction group and the control group without muscone. Compared to the strain with the original receptor STE2 not knocked out, the background signal noise in the glucose group significantly decreased after the original receptor STE2 was knocked out. The experiment confirmed that knocking out the original receptor STE2 of the Saccharomyces cerevisiae mating pathway reduced the noise of the background mating signal, thus improving the reliability of the muscone molecular switch signal.</p>
<p>Based on the previous experimental results of the muscone molecular switch signal, we designed a knockout system targeting the original receptor STE2 of the mating pathway in <i>Saccharomyces cerevisiae</i>, including the gRNA and Cas9 protein targeting STE2. We performed sequencing on the successfully transformed strains and confirmed that base deletions and frameshift mutations occurred in the original receptor STE2 sequence. We retransformed the strains with successful STE2 knockout with MOR215&Ga-pESC and pFUS1 promoter-GFP-pYES and repeated the previous induction experimental protocol. The results of the muscone analysis switch signal intensity in the knockout <i>Saccharomyces cerevisiae</i> strain were obtained under a confocal microscope.</p>
<p>The experimental results show that under the muscone induction condition in the galactose group, the fluorescence intensity and proportion of the GFP reporter gene remained significantly higher than that of the control group without muscone. In the glucose group, there was still no significant difference in the fluorescence intensity and proportion of the GFP reporter gene between the muscone induction group and the control group without muscone. Compared to the strain with the original receptor STE2 not knocked out, the background signal noise in the glucose group significantly decreased after the original receptor STE2 was knocked out. The experiment confirmed that knocking out the original receptor STE2 of the <i>Saccharomyces cerevisiae</i> mating pathway reduced the noise of the background mating signal, thus improving the reliability of the muscone molecular switch signal.</p>
<pstyle="text-align: center; font-size: 0.9em; margin-top: 10px;">fig 13 Quantitative analysis of muscone molecular switch fluorescence signal of knocking out STE2 strain</p>
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<h3>Whole therapy system</h3>
<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>
<p>After separately verifying the effectiveness of the muscone molecular switch and the lactic acid secretion system in <i>Saccharomyces cerevisiae</i>, 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 <i>Saccharomyces cerevisiae</i>. For details, refer to design. The muscone receptor, the modified Gα protein, and the lactate dehydrogenase were simultaneously introduced into <i>Saccharomyces cerevisiae</i>. 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 <i>Saccharomyces cerevisiae</i> 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 <i>Saccharomyces cerevisiae</i>. 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>
<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 <i>Saccharomyces cerevisiae</i> 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>
