Currently, the treatment of depression has become a new global problem, and the existing treatments are divided into pharmacological management, physiotherapy, psychotherapy, and some other relaxation modalities, but all of them have serious limitations. In drug treatment, tricyclic antidepressants and tetracyclic antidepressants are usually used, which all lead to different degrees of side effects; in physical therapy, the method usually used is MECT, electroconvulsive therapy, but with more serious sequelae, which can easily lead to anxiety, memory loss, dizziness, headache, and other symptoms after treatment; the effect of psychotherapy varies from person to person, but psychotherapy is expensive, which is difficult for ordinary people to afford.

  At present, as social pressure increases, so does the number of depressed patients. In this context, there is an increasing demand for reasonable and efficient depression treatment methods.

  As mentioned in our proposed implementation, the goal of this project is to achieve the construction of a psychoactive probiotic for the treatment of depression by pouring the gene for human-derived Tropine Hydroxylase I (TPH1), which produces 5-HTP (a precursor substance of pentraxin), and the gene for the γ-aminobutyric acid-producing Cristaline Decarboxylase into E. coli nissle 1917. In order to provide a valid proof of concept, the experiments to be performed include testing the expression of 5-HTP and γ-aminobutyric acid, testing to verify the optimal ph and optimal temperature of the engineered strain, and testing the working capacity of whole cells in an environment simulating intestinal digestion.

  In our project, after completing the construction of the transgenic strain, we needed to verify that the strain could successfully express the desired substances, i.e. 5-HTP and γ-aminobutyric acid.

  We first observed the strain fluorescently transfected under a fluorescent microscope.

  The experimental results demonstrated that the engineered strain we constructed could effectively produce GABA and 5-HTP, but because most of the two substances were not secreted outside the bacterium, resulting in far more fragmented organisms than the non-fragmented group. The production of GABA was much higher than that of 5-HTP. We speculate that this may be due to the long synthesis pathway of 5-HTP or the fact that the culture environment provided was not the optimal environment.

  In this regard, we designed experiments to test and verify the optimal ph and optimal temperature of the engineered strain, and also whether the modified strain can still survive normally in the human body environment.

  We cultured the engineered strains in LB liquid medium at different values of temperature and ph conditions, respectively, to find the most suitable temperature and ph values, as well as to verify if they match the environmental conditions in the human intestine so that they can survive.

  The experimental results show that the optimum temperature of the successfully constructed engineering strain is 37 degrees Celsius, and the optimum ph value is about 7 It roughly matches the temperature and ph value in the intestine and can survive and function in the human body.

  Although we verified the two most important factors for the survival of the engineered strains in the intestine, there are also influences such as the rest of the intestinal flora, the presence of gastric and intestinal fluids, and other digestive fluids, so we roughly simulated the intestinal environment. The main simulated conditions were hp=7, the temperature of 37 degrees Celsius, the addition of bile salts and pancreatic juice, incubation in a hypoxic (hypoxic bag used) environment, and ELISA assay GABA expression content (5-HTP expression was not measured in this experimental validation because the amount of 5-HTP was too small)

  It was verified that both control and mock groups could survive in the simulated intestinal environment, and the yield of the mock group was significantly increased compared with the control group.

  At the same time, we found a new problem in the validation; that is, most of the GABA and 5-HTP products are intracellular and cannot be released extracellularly for human uptake, so we designed a lysis system to help the engineered strain to optimize our design further.

  Because of time, we did not test in the previously cultured nissle 1917 engineered bacteria. The chassis microorganism used was bl21, and this engineered strain only has a lysis module.

  The experimental results showed that when the concentration of protocatechuic acid (PCA) in the intestine can be controlled around 10-7 mol, it can ensure the partial lysis and partial survival of the bacterium. The partial lysis of the bacterium can release part of GABA and 5-HTP, while the surviving part of the bacterium can continue to reproduce.

  The results of this experiment proved the feasibility of our constructed protocatechuic acid promoter in the strain; although it was not operated on the basis of the original engineered strain, the experiment can still prove the feasibility of the theory.

  In our project, we were able to construct expression systems for GABA and 5-HTP in the probiotic bacterium nissle1917. We demonstrate here that the method we have constructed for the expression system of GABA and 5-HTP from E. coli nissle1917 holds true. In addition to this, we also quantified the expression using the ELISA method and found that the strain showed higher expression after lysis. In further experiments, to ensure that the engineered strains can function in the small intestine environment, we determined the optimal ph and temperature of the expression system of GABA and 5-HTP strains, and actually simulated their ability to work in the intestinal digestive environment in detecting whole cells, which proved that our system can be effectively used in the human small intestine environment. Meanwhile, during the validation process, we further optimized the engineered strain for the identified problems. We designed and constructed a lysis system for the strain and experimentally demonstrated the feasibility of the pro-catecholate promoter-dominated lysis system in the strain.

  The proof of concept demonstrates the capability of our system in a way that can be relied upon to optimize the strain prior toa its in vivo implementation. I hope that our approach and efficient expression system can contribute to the treatment of depression.