We have decided to design an affordable, promising, and efficient microbial therapy for IBD, called Muscure. To comprehensively and effectively promote the development of Muscure, we follow the engineering cycle recommended by iGEM at each stage of our work. Each engineering cycle consists of four interconnected and sequentially progressing steps: design, build, test, and learn. In this way, with well-organized division of labor and cooperation, we have achieved rapid iteration of the project. In many cases, we have been able to effectively solve the difficulties we encounter and obtain the results we initially expected for the design.
In therapy system, we have incorporated muscone-sensing receptors, derived from mouse olfactory epithelial cells, into Saccharomyces cerevisiae. These receptors, which are G protein-coupled receptors (GPCR) in eukaryotic cells, have been integrated into the yeast's signaling pathways. By altering the mating pathway of Saccharomyces cerevisiae, we enabled the muscone receptors to function within this microbial chassis. Additionally, we introduced lactate dehydrogenase downstream of the modified mating pathway, thereby redirecting the yeast's anaerobic metabolism to produce lactate, which is intended for the treatment of Inflammatory Bowel Disease (IBD).
To better construct the system of muscone-induced lactate secretion, we split it into two parts: one is lactate secretion, and the other is the muscone switch. We merged the two parts together after confirming that both parts can work normally. This project is divided into three main cycles.
Lactate is the key effector molecule of our treatment project, so it is very important to construct yeast that can secrete lactate normally.
After literature research, we decided to introduce exogenous lactate dehydrogenase (LDH) into yeast cells as an alternative branch in the normal glycolytic process.
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.
fig 1 GAL1 promoter-ldhA-pYES2 plasmid
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. 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.
fig 2 Lactate secretion induction experiment (wt: wild-type yeast; ldhA: transformed ldhA plasmid yeast; gal: induced by galactose; glc: induced by glucose)
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 population density differences between different groups and the induction time are not strictly controlled. However, it also could not rule out the possibility 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.
We redesigned the induction experiment this time. By diluting based on the differences in yeast concentration between the groups after the hunger and induction, We strictly controlled the population density 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.
Same as cycle1.
According to the new experimental design, we conducted the induction experiment again, and the results are as follows. Carbon sources can significantly affect the lactate secretion rate of yeast. In groups induced by galactose, after the induction is complete, using glucose for feeding results in a lactate secretion rate that is much higher than that of galactose. And It can be found that this time, the transformed yeast induced by glucose only has a very low level of lactate secretion.
fig 3 Lactate-induced secretion experiment with a time gradient
fig 4 Corrected lactate secretion induction experiment (wt: wild-type yeast; ldhA: transformed ldhA plasmid yeast; gal: induced by galactose; glc: induced by glucose)
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.
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. More importantly, to address the current medication challenges faced by IBD patients, the upstream drug delivery switch we designed needs to avoid oral administration whenever possible and should be easy to operate. This will provide patients with a better medication experience. After literature investigation, we finally decided to use the muscone receptor as a candidate for the molecular switch which response of diffusible gas muscone. We then need to verify that the muscone receptor can function effectively in yeast cells.
According to Benjamin M Scott team's research, muscone receptors can be directly integrated into yeast cells to function. More specifically, the muscone receptor, as a GPCR, can share downstream signaling pathways with the mating pathway of yeast. This has brought great convenience to our work. Since this receptor is a mouse-derived GPCR, to improve the efficiency of signal transmission, we replaced the C-terminal five amino acids of the Gα protein in the original mating pathway. For more descriptions, please see description.
After confirming the sequence, we placed the muscone receptor and the corresponding Gα protein together under a galactose-induced bidirectional promoter, which can reduce the number of plasmids that need to be transferred in subsequent operations. We also designed a GFP expression plasmid regulated by the downstream response promoter pFUS1 as a identification marker.
fig 5 MOR215&Ga-pESC plasmid
fig 6 pFUS1 promoter-GFP-pYES plasmid
We induced the double-transformed yeast with galactose to express the muscone receptor and the corresponding Gα protein. Then, we induced muscone and observed the expression of GFP fluorescence signal in yeast cells under a confocal microscope. The results are as follows. We can find that in the group expressing the muscone receptor, adding muscone can significantly increase the fluorescence expression of yeast cells. However, what is frustrating is that in the group induced by glucose, no matter whether muscone is added or not, yeast has a very high fluorescence level.
fig 7 Muscone induction experiment (Gal: induced by galactose; Glc: induced by glucose; Mus: induced by muscone)
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.
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 essentially, and our receptor molecular switch can have better robustness and specificity.
We designed a CRISPR knockout plasmid according to the receptor sequence, as shown in the figure below. We transferred the knockout plasmid into wild-type yeast, picked several single clones, and then sequenced the knockout target gene. We selected a strain with a large fragment deletion and frameshift mutation for subsequent experiments.
fig 8 STE2 gRNA&Cas9-pML107 plasmid
We performed plasmid double transformation on the mating pathway knockout yeast strain and wild-type yeast (control), and then conducted the muscone induction experiment again. The results are as follows. We found that compared with the wild type, the nonspecific fluorescence signal was significantly weakened in the yeast strain with mating pathway knockout after induction with glucose.
fig 9 Corrected muscone induction experiment (Ko2: selected one of the CRISPR knockout strains; Gal: induced by galactose; Glc: induced by glucose; +: induced by muscone; -: no special treatment)
Although the experimental results are in line with our expectations, unfortunately, knocking out the mating pathway in yeast itself cannot completely remove the nonspecific noise. Moreover, what is more confusing is that the specific signal of the group induced by galactose is reduced after gene knockout. These may be due to differences in promoter efficiency. We tried to explain and solve this problem, but due to the time limit of the iGEM competition, we did not achieve good results.
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.
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.
We transferred the complete treatment system into mating pathway knockout yeast, and then proceeded to the next step of induction experiment.
fig 10 pFUS1 promoter-ldhA-pYES2 plasmid
We conducted muscone induction on the transferred yeast strain constructed above under glucose or galactose as the carbon source, and then measured the lactate concentration using standard methods. The results are shown below. We found that after knocking out the mating pathway, under the galactose induction, muscone can significantly increase the lactate secretion of yeast cells. However, the glucose induction group has a rather high non-specific lactate secretion.
fig 11 Muscone-induced lactate measurement results of the treatment system. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)
This measurement of lactate secretion further confirms our previous results. The muscone receptor can efficiently activate downstream signals. However, as discussed earlier, the influence of the carbon source still exists. Glucose, as a preferred carbon source for yeast, can amplify the weak leaky expression of lactate dehydrogenase in yeast cells. The final result is that the glucose-induced group shows a higher level of nonspecific expression. To address the impacts caused by different carbon sources, we have decided to change the promoter of the muscone receptor from a galactose-inducible promoter to a constitutive promoter.
To better construct a subsystem that allows Saccharomyces cerevisiae to colonize IBD lesions, we divided it into two sub-parts: one part is the IBD signaling molecule receptor, and the other part is the adhesion protein. Only after confirming that both parts are functioning properly will we combine these two sub-parts. This section is divided into two main cycles.
To enable Saccharomyces cerevisiae to “sense” the presence of IBD, we need a receptor for IBD.
After conducting a literature review, we decided to introduce the tetrathionate sensor TtrSR into yeast cells to serve as a receptor for IBD.
After confirming the sequence, we designed plasmids that can construct the tetrathionate sensor TtrSR system within Saccharomyces cerevisiae, each carrying the URA3 and HIS3 genes as selection marers, as shown in the figure below.
fig 12 pESC-SV40-ttrR-PttrB-EGFP plasmid
fig 13 pYES2-SV40-ttrS plasmid
We induced the transformed yeast with 1mM K2O6 S4 or without K2O6 S4 (as a control) and measured the expression level of EGFP after 12 hours of induction. The final results showed that after constructing the tetrathionate sensor TtrSR system in yeast cells, the cells were able to sense the IBD signal tetrathionate and activate the expression of downstream genes. However, compared to the control group, there was no significant increase in the expression level of the downstream protein. Although the downstream protein was expressed in response to the IBD signal, the expression level still needs to be improved.
fig 14 Tetrathionate induction experiment (s: control group without inducer; s+: K2O6 S4 added)
Regarding this phenomenon, after discussion, we hypothesize that it may be due to the low expression levels of TtrS and TtrR, or it could be that the membrane protein TtrS, originally from prokaryotic cells, is unable to effectively localize and anchor to the membrane after being expressed in yeast cells. We need to further optimize the relevant sequences.
To address the issue of insufficient downstream protein expression, we decided to optimize the relevant sequences to improve the efficiency of the tetrathionate sensor TtrSR system.
After reviewing relevant literature, we further optimized the codons of TtrS and TtrR to enable more efficient expression in yeast cells. Additionally, we optimized TtrS by adding signal peptides and improving the transmembrane sequences.
fig 15
We conducted predictions for the optimized TtrS, including signal peptide cleavage predictions and intracellular membrane localization predictions.
fig 16
After optimization, the localization and anchoring of TtrS showed some improvement. To further test the system, we need to conduct additional tests on the inducer concentration and induction time.
To enable Saccharomyces cerevisiae to colonize the corresponding site after "sensing" the presence of IBD, we used an adhesion protein to achieve this function.
After conducting a literature review, we decided to express the adhesion protein Als3 from Candida albicans in yeast cells to achieve adhesion to intestinal epithelial cells.
After conducting a literature review, we decided to express the adhesion protein Als3 from *Candida albicans* in yeast cells to achieve adhesion to intestinal epithelial cells.
fig 17 pESC-SV40-ALS3 -EGFP plasmid
We conducted an adhesion assay experiment to assess the ability of the transformed yeast to adhere to colon tissue. The final results showed that after expressing Als3 in yeast cells, their adhesion ability to colon tissue significantly improved. However, upon observing the fluorescence expression in Saccharomyces cerevisiae, we found that the fluorescence expression was relatively weak, with many cells not exhibiting strong fluorescence.
fig 18
Regarding this phenomenon, upon further observation of the relevant slides, we found that *Saccharomyces cerevisiae* on the tissue could be effectively distinguished and counted under an optical microscope. To more accurately characterize the adhesion ability of Saccharomyces cerevisiae, we need to further optimize the observation methods.
Based on the observations under the optical microscope, we plan to repeat the adhesion assay experiment while improving the observation and counting methods for Saccharomyces cerevisiae.
We will continue using the same yeast strains and procedures as before but modify the observation techniques.
We re-conducted the counting and analysis, with the relevant results shown in the figure below. As can be seen, we were still able to demonstrate that after expressing Als3, the colonization ability of Saccharomyces cerevisiae showed a significant improvement.
fig 19
With the improved observation techniques, we can now more accurately characterize the adhesion ability of *Saccharomyces cerevisiae*. Moving forward, we can perform in vivo experiments or use IBD-affected tissues to assess the adhesion ability, allowing for a more comprehensive evaluation of the adhesion capacity of the modified Saccharomyces cerevisiae.