Difference between revisions of "Team:NCKU Tainan/Proof Of Concept"

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

The biggest challenge to Menbles, our bubble, is the digestion of food in the stomach. Because most food coming from our mouth is digested in the stomach by the presence of a very strong acid called gastric acid, Menbles must be ensured to withstand the conditions in our stomach, due to our product’s aim to release the bacteria in the intestine, where our bacteria is supposed to produce taurine and then transported to the brain via the gut-brain axis. Luckily, sodium alginate is able to withstand these extreme conditions in the stomach. Sodium alginate itself, has been proven to have very little effect on itself when under extreme stomach conditions[5]. Therefore, we believe that it is a perfect solution for our case, for it to encapsulate our bacteria and not release it until reaching the intestine, where combined action of acid and trypsin can break the structure of alginate down[1] and releasing the bacteria.

However, another challenge we encountered is that although alginate performs very well under acidic conditions, it does not, however, perform too well under basic conditions[2]. Therefore, we tested our bubbles containing E. coli Nissle 1917 to be submerged into phosphate buffers of four different pHs, reflecting the range of pHs of the milk tea drink. We also tested different bubble sizes to reflect the difficulty of swallowing among patients of mental illnesses[4], which themselves cannot chew our bubble to prevent early release of bacteria. Because of this, we are interested to make our bubble as small as possible, so that these people will not feel the bubble as they are being swallowed. As a result, we also conducted experiments using different sizes of bubbles to identify which bubble size has the most stable recovery rate that we can use for our final product. Below are the results of the rate of recovery of our bubble for the 14.14 mm3 (Large) bubble (Fig. 1), 4.19 mm3 (Medium) bubble (Fig. 2), and 1.77 mm3 (Small) bubble (Fig. 3).

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%.

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.

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.