<p>Saccharomyces cerevisiae is a premier chassis organism for our project. As a kind of common yeast found in human gut, Saccharomyces cerevisiae is considered safe for human and has been extensively studied for its potential application in synthetic biology. As a eukaryotic organism, it possesses more complex and precise mechanisms for gene expression regulation, and various modification approaches also enhance its ability to secrete target proteins. Of note, it has been employed to prevent and treat various diarrheal disorders due to its capability to reshape the balance of gut flora. We believe that Saccharomyces cerevisiae is the best chassis organism to achieve our goal.</p>
<h3>Circuit</h3>
<p>In order to develop a biological therapy for IBD, two fundamental aspects are taken into our consider: controlled release of drug and targeting.</p>
<p>We hope to provide a non-invasive and gentle drug delivery method for IBD patients. Therefore, we designed muscone as a molecular switch for the drug delivery engineering strain. We transferred the muscone receptor from mouse olfactory epithelial cells into Saccharomyces cerevisiae. By modifying the mating pathway of Saccharomyces cerevisiae, we enabled the muscone receptor to function as a signal switch in the yeast.</p>
<p>We assisted the muscone receptor in functioning as a signal switch in Saccharomyces cerevisiae through the G protein-coupled pathway by introducing a Gα protein modified with 5 amino acids at its C-terminus. We also linked the lactate dehydrogenase gene downstream of the promoter in the mating pathway. In the presence of muscone molecules, the molecular switch is activated, and the expression of lactate dehydrogenase is initiated through the mating pathway of the G protein-coupled Saccharomyces cerevisiae.</p>
<p>We altered the anaerobic metabolic pathway of Saccharomyces cerevisiae by introducing lactate dehydrogenase, enabling it to produce D-lactate and secrete it into the surrounding environment for the purpose of inhibiting the abnormal activation of immune cells in the intestine, thereby alleviating the symptoms of IBD patients.</p>
<p>To eliminate the signal interference from the natural mating process of Saccharomyces cerevisiae, we knocked out the original mating receptor STE2 in the yeast. Through experimentation, we confirmed that the knockout reduced the background noise signals in the wild-type yeast, thereby enhancing the stability of the system. In the experiment, we adopted a strategy of separately testing the upstream and downstream components of the system before integrating and validating them. First, we used the GFP reporter gene instead of lactate dehydrogenase to test whether the muscone molecular switch introduced into Saccharomyces cerevisiae could function properly. Meanwhile, we used the galactose promoter to express lactate dehydrogenase, verifying whether lactate dehydrogenase could alter the anaerobic metabolic pathway of Saccharomyces cerevisiae and successfully secrete D-lactate. After that, we synthesized the complete biological system and validated that under the muscone signal, the yeast could synthesize and secrete D-lactate.</p>
<p>For more details, see the <ahref="https://2024.igem.wiki/Tsinghua/therapy-system"style="color: #FF5151">Therapy system</a>.</p>
