<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>
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<h4>Cycle2</h4>
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<h5>Design</h5>
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<h5>Learn</h5>
<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>
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<h4>Cycle2</h4>
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<h5>Design</h5>
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<h5>Learn</h5>
<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>
