Sunnywu0369 (Talk | contribs) |
Sunnywu0369 (Talk | contribs) |
||
Line 39: | Line 39: | ||
<h2 id ="Subtitle1">1. Bubble Production</h2> | <h2 id ="Subtitle1">1. Bubble Production</h2> | ||
− | + | <p> After we consume the engineered E. coli Nissle bubbles, the number of probiotics actually remaining in the human body is always confusing us. As a result, we intended to take the retention rate of E. coli Nissle in the jejunum into consideration. Nevertheless, due to the restriction of human trials and ethical issues, we came up with an idea to produce f(int) - a microfluidic channel to simulate the jejunum environment.</p> | |
+ | <p> In our generation 1 experimental results , as shown in Figure 1, we found that the retention rate of microfluidic chip w/ CF+2villi is higher than any other channel. In addition, when we compare the result of the same channel with additional 5% HA (Hyaluronic acid) that will be the substitution of mucus, the retention rate increases by 5.45%.</p> | ||
+ | |||
+ | |||
+ | <div class="container-fluid p-0"> | ||
+ | |||
<div class="row no-gutters"> | <div class="row no-gutters"> | ||
− | + | <div class="col-lg "> | |
− | + | <figure class="d-flex flex-column justify-content-center align-items-center px-lg-3"> | |
− | + | <a href="https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg" target="_blank" style="width:50%"><img src="https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg" alt="" title="" style="width:100%"></a> | |
− | + | <figcaption class="mt-3">Fig. 4. The retention rate of different microfluidic channels. (w/ CF (structure contains circular folds); w/ CF+villus means (structure contains villus per circular fold); w/ CF+2villi (structure contains 2villi per circular fold) )</figcaption> | |
− | + | </figure> | |
− | + | </div> | |
− | + | </div> | |
+ | </div> | ||
+ | <p> The approach that we took to use 5% HA is because Jejunum intestinal juice has 0.2 to 5% Mucin(MUC2)[1], and HA was used because of its same sticky characteristic with Mucin and due to its low availability. Our experiment result shows that the microfluidic channel with a complex structure such as channel w/ CF+2villi that includes additional Hyaluronic acid is the most suitable for simulating the human intestine.</p> | ||
+ | |||
+ | |||
</section> | </section> | ||
Line 62: | Line 71: | ||
<!-- put link in href & src , width in a can adjust--> | <!-- put link in href & src , width in a can adjust--> | ||
<a href="https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg" target="_blank" style="width:90%"><img src="https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg" alt="" title="" style="width:80%;display:block; margin:auto;" ></a> | <a href="https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg" target="_blank" style="width:90%"><img src="https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg" alt="" title="" style="width:80%;display:block; margin:auto;" ></a> | ||
− | <figcaption>Fig. | + | <figcaption>Fig. 4. The retention rate of different microfluidic channels. (w/ CF (structure contains circular folds); w/ CF+villus means (structure contains villus per circular fold); w/ CF+2villi (structure contains 2villi per circular fold) )</figcaption> |
</figure> | </figure> | ||
</div> | </div> |
Revision as of 11:09, 17 October 2021
![](https://static.igem.org/mediawiki/2021/f/fa/T--NCKU_Tainan--proof.png)
The overall goal of MenTAUR is to relieve depressive symptoms by increasing taurine levels in the intestine. To achieve this, we developed multiple models and conducted wet-lab experiments to prove that our bubble, Menbles, can effectively function and release taurine in the human intestine. Menbles is designed to withstand the acidic environment of the stomach, protecting our encapsulated bioengineered E. coli. We developed a microfluidic chip, f(int), that simulates the intestinal environment, allowing us to determine the retention of E. coli in the jejunum. After confirming that most of our E. coli can adhere to the intestinal walls, we proved that the E. coli could detect stress biomarkers reactive oxygen species (ROS) and IFN-γ through the oxidative stress and IFN-γ sensing systems and produce the taurine production enzymes. Finally, with math-based calculations, we can model the mechanism through which taurine converts into Tau-Cl and reduces ROS and IFN-γ levels in the body, relieving depressive symptoms through the gut-brain axis.
1. Bubble Production
After we consume the engineered E. coli Nissle bubbles, the number of probiotics actually remaining in the human body is always confusing us. As a result, we intended to take the retention rate of E. coli Nissle in the jejunum into consideration. Nevertheless, due to the restriction of human trials and ethical issues, we came up with an idea to produce f(int) - a microfluidic channel to simulate the jejunum environment.
In our generation 1 experimental results , as shown in Figure 1, we found that the retention rate of microfluidic chip w/ CF+2villi is higher than any other channel. In addition, when we compare the result of the same channel with additional 5% HA (Hyaluronic acid) that will be the substitution of mucus, the retention rate increases by 5.45%.
The approach that we took to use 5% HA is because Jejunum intestinal juice has 0.2 to 5% Mucin(MUC2)[1], and HA was used because of its same sticky characteristic with Mucin and due to its low availability. Our experiment result shows that the microfluidic channel with a complex structure such as channel w/ CF+2villi that includes additional Hyaluronic acid is the most suitable for simulating the human intestine.
2. Microfluidic Device
After we consume the engineered E. coli Nissle bubbles, the number of probiotics actually remaining in the human body is always confusing us. As a result, we intended to take the retention rate of E. coli Nissle in the jejunum into consideration. Nevertheless, due to the restriction of human trials and ethical issues, we came up with an idea to produce f(int) - a microfluidic channel to simulate the jejunum environment.
In our generation 1 experimental results , as shown in Figure 1, we found that the retention rate of microfluidic chip w/ CF+2villi is higher than any other channel. In addition, when we compare the result of the same channel with additional 5% HA (Hyaluronic acid) that will be the substitution of mucus, the retention rate increases by 5.45%.
![](https://static.igem.org/mediawiki/2021/f/fd/T--NCKU_Tainan--G1-R.jpg)
3. Taurine Production Experiments
After confirming that most of the engineered bacteria can adhere to the walls of the intestine, we must prove that the bacteria can produce taurine. We designed our engineered bacteria to produce these enzymes only when the body is under high-stress levels, which are signaled by high levels of reactive oxygen species (ROS) and IFN-γ. The following experiments were performed to confirm that both oxidative stress and IFN-γ sensing systems can effectively detect their respective stress biomarkers and initiate taurine production.
As seen in Fig. 5, Δcan::CmR and Δcan::FRT requires a higher CO2 level to survive. In doing so, we have proved that we have successfully knocked out the can gene.
4. Microfluidic Device
To ensure the biosafety of our engineered E. coli Nissle, we did a phenotype test by streaking out the can gene mutant bacteria on different plates and placing them in different conditions.
As seen in Fig. 5, Δcan::CmR and Δcan::FRT requires a higher CO2 level to survive. In doing so, we have proved that we have successfully knocked out the can gene.
![](https://static.igem.org/mediawiki/2021/1/18/T--NCKU_Tainan--backtotop.png)