iGEM 2024 — Team tsinghua


{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

* [Therapy System](#Therapy System)
* [Colonization System](#Colonization System)

0%

---

![ibd_figure]( https://static.igem.wiki/teams/5187/wiki-engineering-fig/engineering.png)

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.

## Therapy System --- 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.

### Big circle1: Lactate secretion

Lactate is the key effector molecule of our treatment project, so it is very important to construct yeast that can secrete lactate normally.

#### Cycle1

---

##### Design

After literature research, we decided to introduce exogenous lactate dehydrogenase (LDH) into yeast cells as an alternative branch in the normal glycolytic process.

##### Build

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure1.png)

Fig 1 GAL1 promoter-ldhA-pYES2 plasmid

##### Test

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure2.png)

Fig 2 Lactate secretion induction experiment (wt: wild-type yeast; ldhA: transformed ldhA plasmid yeast; gal: induced by galactose; glc: induced by glucose)

##### Learn

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.

#### Cycle2

---

##### Design

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.

##### Build

Same as cycle1.

##### Test

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure17.png)

Fig 3 Lactate-induced secretion experiment with a time gradient

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure3.png)

Fig 4 Corrected lactate secretion induction experiment (wt: wild-type yeast; ldhA: transformed ldhA plasmid yeast; gal: induced by galactose; glc: induced by glucose)

##### Learn

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.

### Big circle2: Muscone molecular switch

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.

#### Cycle1

---

##### Design

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.

##### Build

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure4.png)

Fig 5 MOR215&Ga-pESC plasmid

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure5.png)

Fig 6 pFUS1 promoter-GFP-pYES plasmid

##### Test

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure6-new.jpg)

Fig 7 Muscone induction experiment (Gal: induced by galactose; Glc: induced by glucose; Mus: induced by muscone)

##### Learn

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.

#### Cycle2

---

##### Design

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.

##### Build

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure7.png)

Fig 8 STE2 gRNA&Cas9-pML107 plasmid

##### Test

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure8.jpg)

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)

##### Learn

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.

### Big circle3: Whole system

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.

#### Cycle1

---

##### Design

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.

##### Build

We transferred the complete treatment system into mating pathway knockout yeast, and then proceeded to the next step of induction experiment.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure9.png)

Fig 10 pFUS1 promoter-ldhA-pYES2 plasmid

##### Test

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.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure18.png)

Fig 11 Muscone-induced lactate measurement results of the treatment system. (gal: induced by galactose; glc: induced by glucose; mus: induced by muscone)

##### Learn

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.

## Colonization System --- In order to better construct the colonization system that enables *Saccharomyces cerevisiae* to colonize IBD lesions, we have divided it into two sub-parts: one is the IBD signaling molecule sensor, and the other is the adhesion protein. Only after both parts are confirmed to be functioning properly, we will integrate these two sub-parts together. This part is divided into two main loops.

### Big circle1: Tetrathionate sensor

To enable *Saccharomyces cerevisiae* to “sense” the presence of IBD, we need a sensor for IBD. Through literature research, we have found that the concentration of tetrathionate in the intestine can characterize the degree of IBD. Additionally, there is current research that has constructed a tetrathionate sensor TtrSR in E. coli. Therefore, we have decided to introduce the tetrathionate sensor TtrSR into yeast cells, using it as a sensor for IBD.

#### Cycle1

---

##### Design

We have decided to introduce the tetrathionate sensor TtrSR into *Saccharomyces cerevisiae* cells, utilizing it as a detector for IBD. To test its effectiveness within the cell, we will express EGFP downstream and measure its expression level through fluorescence intensity.

##### Build

After confirming the sequence, we designed plasmids pECS and pYES2 that can be used to construct the tetrathionate sensor TtrSR system in brewing yeast. These plasmids carry the URA3 and HIS3 genes, respectively, as selection markers. figures below show the details.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure11.png)

Fig 12 pESC-SV40-ttrR-PttrB-EGFP plasmid

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure12.png)

Fig 13 pYES2-SV40-ttrS plasmid

##### Test

We induced the transformed yeast with 1 mM K2O6S4 or without K2O6S4 (control) and measured the expression of EGFP at 0.5, 1, 2, 4, 8, 12, and 24 hours post-induction. However, the final results from the fluorescence confocal microscopy showed no fluorescence in either the experimental or control groups.

##### Learn

Regarding this phenomenon, we speculate that it might be due to the fact that the samples were not immediately prepared and observed after being collected at different time points. Instead, they were all collected first and then prepared for observation after some time had passed, which could have led to cell death, protein degradation, or fluorescence quenching. It is also possible that the induction conditions were not suitable. Therefore, we need to modify the protocol and experimental plan.

#### Cycle2

---

##### Design

To address the issue of not being able to observe fluorescence, we have made revisions to the existing experimental protocol to eliminate interference from unrelated factors on the experimental results.

##### Build

same as cycle 1

##### Test

We increased the concentration of the tetrathionate inducer from 1mM to 2mM and collected the samples immediately after 24 hours of induction to prepare temporary slides for observation.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-colonization-fig/pic11.png)

Fig 14 Statistical results of tetrathionate induction experiment (s: control group without inducer; s+: K2O6S4 added)

##### Learn

After optimizing the protocol, we successfully observed green fluorescence. The experimental results showed that the expression of the downstream protein EGFP increased after tetrathionate induction, but there was no significant difference compared to the control group. In the S+ group, although EGFP was expressed, the fluorescence was weak and the expression level was low.

We speculate that this may be due to compatibility issues after the tetrathionate sensor TtrSR was transferred from E. coli to *Saccharomyces cerevisiae*. Due to differences in protein expression and delivery systems between eukaryotic and prokaryotic cells, TtrS may not effectively realize its function in *Saccharomyces cerevisiae* due to issues such as signal peptides and transmembrane domains, and may not be properly localized to the cell membrane. Additionally, the expression levels of TtrS and TtrR may be insufficient in brewing yeast. In subsequent experiments, we plan to optimize TtrS and TtrR, including codon optimization, signal peptide prediction and optimization, membrane localization prediction, and transmembrane domain optimization, in order to enable TtrRS to function better in brewing yeast.

### Big circle2: Adhesion protein

To enable *Saccharomyces cerevisiae* to “sense” the presence of IBD and colonize the corresponding sites, we utilize an adhesion molecule for this function. Through literature research, we have found that the adhesion protein Als3 from Candida albicans can bind to E-cadherin on epithelial cells, thereby achieving adhesion. Since *Saccharomyces cerevisiae* and Candida albicans are both common fungi in the human body and share some similarities, we have decided to express Als3 in *Saccharomyces cerevisiae* to achieve adhesion to intestinal epithelial cells.

#### Cycle1

---

##### Design

We have decided to express both Als3 and EGFP in yeast cells and to test the function of Als3 in brewing yeast through adhesion assay experiments.

##### Build

After confirming the sequence, we designed a plasmid that can express Als3 in brewing yeast, using EGFP as a tracing marker. Additionally, this plasmid carries URA3 as a selection marker. Figure below shows the details.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-engineering-fig/figure16.png)

Fig 15 pESC-SV40-ALS3 -EGFP plasmid

##### Test

We conducted an adhesion ability assay using transformed *Saccharomyces cerevisiae* on small intestinal tissue. The final results indicate that after expressing Als3 in yeast cells, the adhesion ability of these cells to small intestinal tissue has improved to some extent. However, there is no significant advantage compared to the WT (wild-type) cells.

![ibd_figure](https://static.igem.wiki/teams/5187/wiki-colonization-fig/pic13.png)

Fig 16 **Statistical results of quantification of attachment experiment** (Als3: the attachment of *saccharomyces cerevisiae* expressing Als3; WT: the attachment of wild-type *saccharomyces cerevisiae*, control groups)

##### Learn

The experimental results indicate that expressing Als3 can enhance adhesion ability to some extent. In order to explore better solutions, we need to test the adhesion capabilities of more adhesion molecules or systems. We plan to introduce CSP or PfEMP1 from Plasmodium, surface glycoproteins from Giardia lamblia, or surface antigen SAG1 into *Saccharomyces cerevisiae* , and compare their adhesion effects after expression in *Saccharomyces cerevisiae*.