<p>Lactate is the key effector molecule of our treatment project, so it is very important to construct yeast that can secrete lactate normally.</p>
<h4>Cycle1</h4>
<hr>
<h5>Design</h5>
<p>After literature research, we decided to introduce exogenous lactate dehydrogenase (LDH) into yeast cells as an alternative branch in the normal glycolytic process.</p>
<h5>Build</h5>
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<p>For this abnormal phenomenon, after discussion, we speculated that it might be that the ldhA gene on the plasmid has background expression, and yeast grows faster in the carbon source environment of glucose, so it can compensate for the deficiency in protein expression level in terms of cell number. The comprehensive result is that the transformed yeast induced by glucose also has the same level of lactate secretion as the yeast induced by galactose. Our induction scheme design this time is slightly rough, and the OD differences between different groups and the induction time are not strictly controlled. However, it is also impossible to rule out that the background expression is very strong, even covering the gain brought by galactose induction. We need to design a more refined experiment to verify.</p>
<hr>
<h4>Cycle2</h4>
<hr>
<h5>Design</h5>
<p>We redesigned the induction experiment this time, strictly controlling the OD differences between different groups of yeast and the induction time, and the results are as follows. In addition, we also took samples with a time gradient during the induction process for more detailed analysis, please see wet lab for details.</p>
<h5>Build</h5>
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<h3>Big circle2: muscone molecular switch</h3>
<p>The lactate secretion experiment has confirmed the feasibility of our treatment project. Next, we need to find a molecular switch that matches it. As a basic structure in our design, the molecular switch plays a crucial role. It is through the switch that we can control the timing and quantity of administration to patients.</p>
<h4>Cycle1</h4>
<hr>
<h5>Design</h5>
<p>After literature investigation, we decided to use the muscone receptor as a candidate for the molecular switch. At the same time, because this receptor is a mouse-derived G protein-coupled receptor, we also paired it with the corresponding Gα protein. For specific descriptions, please see description.</p>
<h5>Build</h5>
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<p>The muscone molecular switch designed in our project utilizes the mating pathway signaling pathway that already exists in yeast. Literature research shows that in wild-type yeast, this signaling pathway will be activated under starvation conditions. In our induction process, there is a 5-6 hour starvation time, which may nonspecifically activate the downstream GFP signal. In the galactose-induced group, the muscone receptor and Gα protein will be expressed, which may compete with the mating pathway that naturally exists in yeast cells, thereby weakening this nonspecific activation. In the glucose group, the expression level of the muscone receptor and Gα protein is low, and this nonspecific activation is more obvious.</p>
<hr>
<h4>Cycle2</h4>
<hr>
<h5>Design</h5>
<p>Since the mating pathway signaling pathway that naturally exists in yeast cells can cause nonspecific activation of the downstream, we plan to knock out the receptor of this pathway according to the results of literature research. This will solve the problem of nonspecific expression from the root, and our receptor molecular switch can have better robustness and specificity.</p>
<h5>Build</h5>
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<h3>Big circle3: Integration</h3>
<p>After confirming that both the lactate secretion subsystem and the muscone switch subsystem can work normally, we are ready to integrate the two parts together to construct a complete treatment system.</p>
<h4>Cycle1</h4>
<hr>
<h5>Design</h5>
<p>The molecular switch still uses the muscone receptor and Gα protein mentioned earlier, and the downstream response module is replaced with the lactate dehydrogenase gene regulated by the pFUS1 promoter.</p>
<h5>Build</h5>
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<p>This measurement of lactate secretion further confirms our previous judgment: the mating pathway signaling pathway that naturally exists in yeast cells can cause nonspecific activation of downstream signals. However, unfortunately, in the group induced by galactose, whether it is wild-type yeast or mating pathway knockout yeast, the amount of lactate secretion did not meet our expectations. We will try to solve this problem in the future.</p>
<hr>
<h4>Cycle2</h4>
<hr>
<h5>Design</h5>
<p>Because the synthesis of muscone receptor and Gα protein induced by galactose is not very stable, and in order to better fit the applicability of the subsequent treatment system, we decided to change the promoter of muscone receptor and Gα protein to a strong constitutive promoter in yeast, expecting this to solve the difficulties we encountered earlier.</p>
