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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


To better construct the subsystem of muscone-induced lactate secretion, we split it into two parts: one is lactate secretion, and the other is the muscone switch. We only 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.

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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. 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.

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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 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.

Cycle2


Design

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.

Build

Same as cycle1.

Test

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.

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fig 3 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, and the promoter of the receptor will also use the constitutive promoter in yeast.

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.

Cycle1


Design

After literature investigation, we decided to use the muscone receptor as a candidate for the molecular switch. At the same time, because this receptor is a mouse-derived G protein-coupled receptor, we also paired it with the corresponding Gα protein. For specific 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 downstream identification marker.

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fig 4 MOR215&Ga-pESC plasmid

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fig 5 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 statistics are as follows. We can find that in the group expressing the muscone receptor, we observed that 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.

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fig 6 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 from the root, 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.

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fig 7 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.

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fig 8 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. We tried to explain and solve this problem, but due to the time limit of the iGEM competition, we did not achieve good results. For more details, please see wet lab.

Big circle3: Integration

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 both mating pathway knockout yeast and wild-type yeast (control), and then proceeded to the next step of induction experiment.

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fig 9 pFUS1 promoter-ldhA-pYES2 plasmid

Test

We conducted muscone induction on the two yeast strains constructed above under glucose 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, the nonspecific signal of the treatment system was significantly reduced.

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fig 10 Muscone-induced lactate measurement results of the treatment system. (wt: wild-type yeast transformed with the treatment system; ko2: mating pathway knockout yeast transformed with the treatment system; mus: induced by muscone)

Learn

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.

Cycle2


Design

Because the synthesis of muscone receptor and Gα protein induced by galactose is not very stable, and in order to better fit the applicability of the subsequent treatment system, we decided to change the promoter of muscone receptor and Gα protein to a strong constitutive promoter in yeast, expecting this to solve the difficulties we encountered earlier.

Build

We redesigned the plasmid according to the sequence, as shown in the figure below.

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fig 11

Test

To be continue…

Learn

To be continue…

Colonization System


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.

Big circle1: Tetrathionate sensor

To enable Saccharomyces cerevisiae to “sense” the presence of IBD, we need a receptor for IBD.

Cycle1


Design

After conducting a literature review, we decided to introduce the tetrathionate sensor TtrSR into yeast cells to serve as a receptor for IBD.

Build

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.

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fig 11 pESC-SV40-ttrR-PttrB-EGFP plasmid

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

Test

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.

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fig 13 Tetrathionate induction experiment (s: control group without inducer; s+: K2O6 S4 added)

Learn

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.

Cycle2


Design

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.

Build

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.

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fig 14

Test

We conducted predictions for the optimized TtrS, including signal peptide cleavage predictions and intracellular membrane localization predictions.

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fig 15

Learn

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.

Big circle2: Adhesion protein

To enable Saccharomyces cerevisiae to colonize the corresponding site after "sensing" the presence of IBD, we used an adhesion protein to achieve this function.

Cycle1


Design

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.

Build

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.

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fig 16 pESC-SV40-ALS3 -EGFP plasmid

Test

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.

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fig 17

Learn

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.

Cycle2


Design

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.

Build

We will continue using the same yeast strains and procedures as before but modify the observation techniques.

Test

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.

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fig 18

Learn

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.

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