Team:The Webb Schools/Results

The_Webb_Schools

Results
Plasmid Construction Result
Figure 1. Plasmid diagram
Figure 2. Sequencing result of the plasmid C from the sequencing company
    The sequencing result (Figure 2) shows that the gene efeB and amilGFP are successfully inserted namely the plasmid C was constructed successfully which was used for the subsequent function tests same as the other 3 plasmids.
Function Test Result
1. Bacteria Growth
Table 1. relative biomass percents of bacteria A and B with different concentrations of H2O2
Figure 3. Histogram of relative biomass of bacteria A and B against different concentrations of H2O2
    Bacteria A contains plasmid A, pUC57-efeB, and the bacteria D contains plasmid D, pUC57. When the concentration of hydrogen peroxide is 0 mM, the relative biomass of bacteria A and bacteria C are the same. As the concentration of hydrogen peroxide increases to 1mM, the relative biomass of bacteria A increases while the relative biomass of bacteria C remains the same, which shows that without efeB, bacteria is unable to grow when ROS is present. As the concentration of hydrogen peroxide increases to 2mM, the relative biomass of bacteria A decreases by half while the relative biomass of bacteria D decreases to 0mM. This shows that bacteria with the efeB gene is able to survive when the level of ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of both bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are unable to survive regardless of the presence of the efeB gene.
Figure 4. Line chart of the OD600 of bacteria A, C and D against hours in 2.5mM H2O2
    The bacteria A contains plasmid A, pUC57-efeB. The bacteria C contains plasmid C, pUC57-efeB-amilGFP. The bacteria D contains plasmid D, pUC57. The bacteria are placed in 2.5mM hydrogen peroxide and their growth is measured for the first 4 hours. Bacteria D, without the efeB gene, has lower growth than bacteria A and bacteria C. This shows that efeB gene helps bacteria to grow when ROS is present.
2. MDA and Fluorescence Measuring
Figure 5. Histogram of MDA in the systems with different H2O2 concentration of bacteria A, C and D
    Bacteria A, C, D were placed in hydrogen peroxide solution for 21 hours and the concentration of MDA was measured. Bacteria A and C kept the MDA concentration dramatically lower than those of Bacteria D under the hydrogen peroxide concentration of 0mM, 2mM and 2.5mM. There is a certain amount of MDA concentration within DH5α when the H2O2 concentration is 0mM. Starting from the original DH5α MDA concentration to the higher MDA concentration imposed by the 2mM and 2.5mM H2O2 concentration, Bacteria A and C with the efeB gene all effectively reduced the MDA concentration to the level lower than Bacteria D.
Figure 6. Histogram of fluorescence intensity of bacteria C system with different H2O2 concentration
    In bacteria C, the expression of GFP is controlled by the expression of efeB. The more intense the fluorescence is, the better efeB will express. H2O2 climbed from 0 to 2.5 mM, and the intensity of fluorescence climbed higher as well. The tendency of the intensity shows that the expression of efeB increases with the H2O2 concentration. Expression of efeB disturbs the production of MDA.
Figure 7. Line chart of MDA in bacteria C system with 2.5mM H2O2 against hours
    Bacteria C was placed in the 2.5nM hydrogen peroxide solution, and the level of MDA was measured under different hours. During the first 4 hours, the concentration of MDA increased up to 20 mmol/mg. The concentration of MDA decreased to 6 mmol/mg for the next four hours. The measured levels of MDA demonstrate that the efeB gene can curb the increase of MDA and keep it in a relative low level.
    In order to deeper analyze the factors which may affect the ability of bacteria C and bacteria A to inhibit MDA production, more function tests would be designed and conducted in the future.