Team:The Webb Schools/Engineering


In modern society, more and more people suffer from mild or severe depression. It has been reported that malondialdehyde (MDA) level in the plasma of depressed patients is significantly increased. After receiving conventional antidepressant treatment, the patient's MDA level decreased to the same as that of healthy people. Therefore, researchers believe that oxidative stress may play an important role in the occurrence and development of depression, and the activity of antidepressant therapy may be mediated by improving oxidative stress/antioxidant function. We attempt to express an antioxidant enzyme system (peroxidase gene efeB) in the cell or on the surface of the engineered probiotic bacteria to inhibit the production of malondialdehyde (MDA) by human cells and prevent cell oxidation from causing health damage to the body. So as to prevent or alleviate the condition of depression.
The oxidation rate of peroxisome increases in proportion to the increase of oxygen tension. Especially in the case of high oxygen concentrations, the oxidation reaction of peroxisomes dominates. This characteristic allows peroxisomes to protect cells from the toxic effects of high concentrations of oxygen. efeB reduces the oxidative stress in the cell by competing with mitochondria for oxygen, and reduces the MDA produced during the oxidation of lipids.
Figure 1. Action and function of efeB in MDA reduction.
Four recombinant plasmids were constructed (plasmid A:pUC57-Pro-efeB; plasmid B:pUC57-amilGFP; plasmid C:pUC57-Pro-efeB-amilGFP) (Figure 1). Plasmid A is used to produce recombinant efeB in probiotic bacteria. Plasmids B and C are used to confirm function of the composite part and expression of efeB, respectively. All the constructs are confirmed by DNA sequencing.
Figure 1. Schematic map of plasmids.
Bacteria A (contains plasmid A, pUC57-efeB), and the bacteria D (contains plasmid D, pUC57 empty vector). Bacteria A, which produces efeB enzyme, can survive in high concentration of hydrogen peroxide (Figure 2).
Figure 2. Histogram of relative biomass of bacteria A and B against different concentrations of H2O2.
Bacteria A and C kept the MDA concentration dramatically lower than those of Bacteria D (transformed with pUC57 vector) under the hydrogen peroxide concentration of 0mM, 2mM and 2.5mM. Bacteria A and C with the efeB gene all effectively reduced the MDA concentration to the level lower than Bacteria D (Figure 3).
Figure 3. Histogram of MDA in the systems with different H2O2 concentration.
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. Bacteria with the efeB gene effectively reduced the MDA concentration to the lower level. Our genetically engineered bacteria are promising in preventing or alleviating the condition of depression.