<p>During our brainstorming process, we realized the mighty potency of synthetic biology in medical treatments, especially in the therapy development of <b>inflammatory bowel disease (IBD)</b>, one of the hardest-to-cure conditions in the world. Thanks to the well-understood genetics and its efficient heterologous protein expression capacity, Saccharomyces cerevisiae appears to be a promising chassis organism for developing biological therapies for IBD. Additionally, various synthetic biology components enable us to design multiple control systems, achieving precise and comprehensive control over the treatment. Viewing IBD treatments from a brand-new aspect, we found that biological therapy obtains tremendous advantages not found in traditional chemical drugs. As a result, we have chosen IBD therapy as our project focus of this year.</p>
<p>During our brainstorming process, we realized the mighty potency of synthetic biology in medical treatments, especially in the therapy development of <b>inflammatory bowel disease (IBD)</b>, one of the hardest-to-cure conditions in the world. Thanks to the well-understood genetics and its efficient heterologous protein expression capacity, <i>Saccharomyces cerevisiae</i> appears to be a promising chassis organism for developing biological therapies for IBD. Additionally, various synthetic biology components enable us to design multiple control systems, achieving precise and comprehensive control over the treatment. Viewing IBD treatments from a brand-new aspect, we found that biological therapy obtains tremendous advantages not found in traditional chemical drugs. As a result, we have chosen IBD therapy as our project focus of this year.</p>
<p>Comprising of Ulcerative Colitis (UC) and Crohn's Disease (CD), IBD is a chronic, incurable disease affecting people of all ages worldwide. According to the Global Burden of Disease (GBD), there was a notable increase of 175,904 individuals diagnosed with IBD from 1990 to 2021. Over 1 million residents in the USA and 2.5 million in Europe are estimated to have IBD, with substantial costs for health care. IBD patients normally suffer from abdominal pain and diarrhea, combined with intermittent fever and various extraintestinal symptoms such as arthritis. Besides, IBD is well-known for its complex and unclear pathogenesis, which also explains why no common and satisfying therapy exists.</p>
<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>
<p><i>Saccharomyces cerevisiae</i> is a premier chassis organism for our project. As a kind of common yeast found in human gut, <i>Saccharomyces cerevisiae</i> 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 <i>Saccharomyces cerevisiae</i> is the best chassis organism to achieve our goal.</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>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 <i>Saccharomyces cerevisiae</i>. By modifying the mating pathway of <i>Saccharomyces cerevisiae</i>, 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 <i>Saccharomyces cerevisiae</i> 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 <i>Saccharomyces cerevisiae</i>.</p>
<p>We altered the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i> 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 <i>Saccharomyces cerevisiae</i>, 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 <i>Saccharomyces cerevisiae</i> could function properly. Meanwhile, we used the galactose promoter to express lactate dehydrogenase, verifying whether lactate dehydrogenase could alter the anaerobic metabolic pathway of <i>Saccharomyces cerevisiae</i> 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>
