Part:BBa_K4300002

Added by LZU-HS-Pro-A

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.

Figure 1 Effect of fluorescent transfection

  To further accurately detect the expression amount, we use ELISA (immunoassay (IA)) technique. ELISA technique is performed by solid phase of antigen or antibody and enzymatic labeling of antigen or antibody, and after adding the substrate of enzyme reaction, the substrate is catalyzed by enzyme to become colored product, and the amount of product is observed for qualitative or quantitative analysis. Therefore, we choose this technique for further detection of expression.

Figure 2 Successful expression of γ-aminobutyric acid in nissle 1917 (The amount of GABA is given in mg/L units and to verify expression, both post-cleavage strains and pre-cleavage strains were used for comparison)

Figure 3 Successful expression of 5-HTP in nissle 1917 (5-HTP is given in mg/L units, and to verify expression, both post-cleavage strains and pre-cleavage strains were used for comparison)

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.

Figure 4 GABA expression at different temperatures, GABA is given in mg/L units, strains were not lysed

Figure 5 Expression of GABA under different ph conditions

 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)

Figure 6 Successful expression of GABA in the simulated intestinal environment

 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,

t7 promoter + strong rbs+GAD+TPH-1+ strong terminator

GABA and 5-HTP composite components are produced

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]