The core of our treatment project revolves around three aspects:
Effectiveness: The therapy system we designed can effectively induce yeast to secrete lactic acid;
Safety: Our engineered yeast can specifically colonize the patient's lesion site and die after leaving the patient's body environment;
Feasibility: Muscone can reach the intestines at a higher concentration through inhalation.
We designed detailed physical and chemical experiments to verify the first two aspects; for the third point, due to the safety restrictions imposed by iGEM on participating teams, we only used theoretical models such as gas molecule diffusion to illustrate it. Unfortunately, the plasmid expression system in yeast is not very stable. In addition, the molecular switch of muscone we designed has a complex interplay with the existing signaling pathways in yeast cells.
Therefore, validating experiments is very difficult, especially considering the time constraints of the iGEM competition. Despite referencing a large amount of papers and materials, most of the data we obtained is still frustrating. It is also very difficult to explain these anomalous data. We have overcome all these obstacles and successfully established a more mature muscone molecular switch and colonization system in the yeast system. This indicates that the concept of inhalation therapy with muscone is a theoretically feasible one.
The muscone molecular switch we introduced from the yeast system is a very novel, simple, and responsive regulatory method. By modifying the downstream response signals, it can be easily applied to the design of other genetic engineering projects.
fig 1 Muscone molecular switch-regulated lactic acid secretion system
We introduced the lactate dehydrogenase gene (ldhA) regulated by a galactose promoter into yeast cells, induced it under different conditions, and measured the lactic acid content in the supernatant. We found that, aside from a small amount of leakage expression of the ldhA gene itself, galactose can very efficiently induce yeast to secrete sufficient lactic acid into the external environment. It is worth noting that because glucose is a better carbon source for yeast compared to galactose, the difference in carbon sources can have some effects on lactic acid secretion beyond just gene expression, which we have not demonstrated here. There is a more detailed discussion in the wet lab section.
fig 2 Results of yeast-induced lactic acid secretion. This image shows the differences in lactic acid secretion by yeast induced by different carbon sources (glucose or galactose).(wt: wild-type yeast, ldhA: yeast transformed with a plasmid containing the ldhA gene)
We simultaneously introduced plasmid vectors expressing the muscone receptor and the corresponding Gα protein into yeast cells, along with a plasmid vector expressing the GFP protein controlled by the pFUS1 promoter. We used galactose to induce the expression of receptors and Gα proteins, and added a certain concentration of muscone for induction. The results are as follows. We found that, aside from the weak background expression, muscone can very efficiently induce the expression of the downstream controlled GFP gene.
fig 3 Yeast specifically expresses GFP through designed pathways in the condition of muscone inducing
We combined the muscone molecular switch with the lactate dehydrogenase gene to measure the differences in the rate of lactic acid secretion by yeast induced by different concentrations of muscone.
fig 4 Thiosulfate-induced Als3-dependent colonization system
We introduced the Als3 gene, regulated by a galactose promoter, into yeast and found that after the addition of galactose, it could attach in greater numbers to human intestinal epithelium compared to wild-type yeast.
We introduced plasmids expressing downstream regulated GFP and thiosulfate receptors into yeast simultaneously and found that thiosulfate can significantly enhance the intensity and proportion of GFP signal expression in yeast.
……to be continue
……to be continue