<p>Fortunately, the leakage expression of the ldhA gene is very low, and the induction of the galactose promoter is also effective. It is only because the difference in carbon sources will affect the reproduction and glycolytic metabolism rate of yeast itself, which in turn affects the overall lactate secretion level. However, these do not affect our project. Later, the promoter of the ldhA gene will be changed to the pFUS1 promoter regulated by the downstream of the yeast mating pathway, and the promoter of the receptor will also use the constitutive promoter in yeast.</p>
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<h3>Big circle2: Lactate secretion</h3>
<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>
<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>
<p>After confirming the sequence, we designed a plasmid containing the lactate dehydrogenase gene (ldhA) driven by the galactose promoter, which has the URA3 gene as a selection marker, as shown in the figure below.</p>
<p>We induced transformed yeast and wild-type yeast (control) with galactose or glucose (control) and measured the lactate content in the supernatant. The final results showed that the plasmid we designed can normally express the LDH protein in yeast cells, and under the induction of galactose, it can secrete a higher concentration of lactate into the supernatant. However, in wild-type yeast, no significant lactate secretion was observed under either galactose or glucose induction. However, what is puzzling is that the transformed yeast induced by glucose also has the same level of lactate secretion.</p>
<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>
<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>
<p>Same as cycle1.</p>
<h5>Test</h5>
<p>According to the new experimental design, we conducted the induction experiment again, and the results are as follows. It can be found that this time, the transformed yeast induced by glucose only has a very low level of lactate secretion.</p>
<p>Fortunately, the leakage expression of the ldhA gene is very low, and the induction of the galactose promoter is also effective. It is only because the difference in carbon sources will affect the reproduction and glycolytic metabolism rate of yeast itself, which in turn affects the overall lactate secretion level. However, these do not affect our project. Later, the promoter of the ldhA gene will be changed to the pFUS1 promoter regulated by the downstream of the yeast mating pathway, and the promoter of the receptor will also use the constitutive promoter in yeast.</p>
