Difference between revisions of "Team:NCKU Tainan/Results"

Menbles Production Experiments

Goal

To test whether our final product (Menbles) can effectively protect E. coli Nissle 1917 inside the bubble under the pH conditions of the milk tea drink, and to compare which bubble size has the best efficiency in protecting our bacteria.

Achievements

1. Proved that the bubble is effective to protect the bacteria inside, maximize recovery, and minimize losing the bacteria before being digested, in the pH conditions of the milk tea drink.

2. Identified which bubble size is the most stable based on recovery rates.

Process

After deciding the raw materials used for our bubble, which are sodium alginate and calcium chloride, we immediately conducted experiments testing the amount of bacteria recoveredy from the bubble itself at different pHs of the milk tea drink. We tested with 4 different pHs, pH 4.5, 5.0, 5.5, and 7.0. The bubble is produced by mixing 2 ml of OD600 1 E. coli Nissle 1917 with 50 ml alginate solution, and 2.5 ml of the solution is dripped drop by drop using different volumes of laboratory pipettes into a calcium chloride solution. As a result, we experimented with 3 different sizes of the bubble, with volumes approximately 14.14 mm3 (Large), 4.19 mm3 (Medium), and 1.77 mm3 (Small).

The bubbles are then separated from the calcium chloride solution using filtration,and the bubbles are equally distributed and submerged into 12.5 ml disodium hydrogen diphosphate buffer of the 4 different pHs for 2 hours in room temperature.

After 2 hours, 60 μl of the supernatant for every pH is spread onto an LB plate medium, used as the supernatant data. Then, the bubbles are separated from the buffer and crushed using a syringe, in which after crushing, the crushed bubbles are then put back into the buffer separated earlier. 60 μl of the supernatant is again spread onto another LB plate medium, which will then be used as the bubble data. A negative control experiment involving just the buffer of different pHs without bubbles submerged in it is also spread onto another LB plate medium, to identify any contamination that could affect the result of the experiment.

The plates are then incubated for 12 hours in 37°C, in which after incubation CFU quantification is conducted to determine the bacteria recovery rate of the bubble. The results below are the bacteria recovery rate of the 14.14 mm3 (Large) bubble (Fig. 2), 4.19 mm3 (Medium) bubble (Fig. 3), and 1.77 mm3 (Small) bubble (Fig. 4). All our negative control plates did not grow any colonies, so the results obtained are devoid of any contamination. To count the percentage of bacteria, we multiplied the CFU number we obtained from the plate, with the dilution of the sample (60 μl/15 ml) and divided by the total number of bacteria that is supposed to be inside. By calculating the amount of bacteria that is supposed to be in the bubbles in each pH buffer, we can approximately calculate the percentage of bacteria in the supernatant after 2 hours, and the amount of bacteria recovered after crushing the bubble.

Oxidative Stress Sensing

pchR

Interferon-gamma Sensing

Achievements:

Taurine Production

This year, we characterized FNR promoter (BBa_K1123000) which is an anaerobic promoter. Originally, we used this promoter to drive the expression of TAL constructs. However, finding that this promoter has a higher expression level of GFP under aerobic conditions, we did not use this promoter for our project. Instead, we used the strong constitutive promoter J23100 as our promoter.

We first cultured DH5α pSB1C3-P_fnr-GFP under aerobic and anaerobic conditions. After 10 hours of incubation, we fixed the E. coli cells in 2% agarose gel and placed it on a glass slide, then place the glass slide under a microscope for image capturing.

As seen in Fig. 20, the fluorescence signal in single E. coli cell after 10 hours of incubation is significantly higher in aerobic conditions than in anaerobic conditions. Literature has reported that GFP requires oxygen molecules to fold properly^[7] before it can emit fluorescence signal. We cannot exclude the possibility that in anaerobic culture, GFP protein is not folding properly and thus affecting the measurement result. However, we can still conclude this promoter is not anaerobic specific. It is recommended that future iGEMers should use other reporter genes that will not be affected by oxygen like luciferase to further characterize this Biobrick.