Difference between revisions of "Team:NCKU Tainan/Proof Of Concept"
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<figcaption>Fig. 4. <i>p</i>-Coumaric acid/OD<sub>600</sub> levels of <i>E. coli</i> Nissle with <i>tyrP</i>+ TAL w/34 in LB with different concentration of tyrosine</figcaption> | <figcaption>Fig. 4. <i>p</i>-Coumaric acid/OD<sub>600</sub> levels of <i>E. coli</i> Nissle with <i>tyrP</i>+ TAL w/34 in LB with different concentration of tyrosine</figcaption> | ||
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| − | <p> | + | <p> The oxidative stress sensing system regulates the production of L-cysteine sulfinic acid decarboxylase (CSAD). It consists of the soxR gene, promoter PsoxS, and the csad gene. When SoxR protein is fully oxidized, it becomes a powerful transcription activator of PsoxS, leading to the expression of the downstream gene. To test the intensity and specificity of the oxidative stress sensing system, the oxidative stress assay was performed.</p> |
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Revision as of 07:38, 17 October 2021
Bubble Production
Menble is a special edible bubble that is a unique depression medication due to its form of coming with a very popular drink all around the world, bubble milk tea. Our depression medication is designed to be incorporated into everyone’s daily lives, in which everyone could easily access our bubble, which means that existing sufferers of depression, who can’t seek medical help due to stigma, can obtain this sweet, delicious drink with a bubble that contains our engineered bacteria to help cure depression by producing taurine.
The biggest challenge to Menble, 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, Menble 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[5] 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[6]. 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.).
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.
The oxidative stress sensing system regulates the production of L-cysteine sulfinic acid decarboxylase (CSAD). It consists of the soxR gene, promoter PsoxS, and the csad gene. When SoxR protein is fully oxidized, it becomes a powerful transcription activator of PsoxS, leading to the expression of the downstream gene. To test the intensity and specificity of the oxidative stress sensing system, the oxidative stress assay was performed.
Model
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.
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.
