Team:NWU-China/Experiments
Experiment
1.Experiment of biosensor
1.1 Bacterial strains and growth conditions
E. coli DH5α cells were used as the hosts for molecular cloning. They were cultivated in LB broth under shaking at 200 rpm at 37℃.
1.2 Measurements of fluorescence intensity
Biosensor strains were grown in LB broth at 37℃ overnight. In a 96-well plate, The cells were diluted (1:50; v%) in 100μl of the M9 (supplemented with 0.4% glucose, 0.24 mg/mL MgSO4, 11.1μg/mL CaCl2, 0.3 μM thiamine hydrochloride, and 50μg/mL kanamycin) to each well, and Phe/Tyr was added at a concentration gradient of 0, 12.5, 25, 50, 100, 200μM. Place the 96-well plate into an automatic microplate reader. Incubate at 37℃ 24h and record the fluorometric value(530 nm/590 nm for RFP, 485 nm/528 nm for GFP) and OD600 for each well every 30 minutes.Each group should be repeated for at least 3 times.
Fig. 1 experimental flow chart
1.3 Experiment of interference of urine components
Since the experiment of collecting human urine involves ethical requirements, we decided to formulate it according to its main content in vitro.
Main ingredient |
Content |
Water |
95% |
Protein |
0% |
Glucose |
0% |
Urea |
1.8% |
Uric acid |
0.05% |
Inorganic salt |
1.1% |
Table 1: Main component of urine * Data From Wikipedia
The components in the urine that affect the experimental results were mainly urea and uric acid, but it was unrealistic to culture cells with urine, so we configured a mother liquor of the M9 medium containing 1.8% urea and 0.05% uric acid. Because the Phe content in the urine of PKU patients was around 20mM (H.A. Harper et al., 1973), we added the engineered bacteria to the M9 medium (with 100μM Phe/Tyr) diluted 200 times urea and uric acid mother liquor, and set a blank control (no urea and urea added) and at 37℃, 200 rpm culture. The samples were taken at 8h, 12h, 16h, 20h, and 24h, and the fluorescence value and OD600 were measured in a microplate reader, and each experiment was repeated three times.
2. Experiment of colorimetric card
Due to time issues, we only completed in vitro experiments in the color card section. We overexpressed the pigment proteins - amilCP, fwYellow, gfasPurple and spisPink in LB medium with E. coli, extracted and purified, and diluted to the same concentration. In a 96-well plate, the total volume of the pigment protein solution per well was 220μl, fwYellow was added according to a volume gradient of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220μl, and then amilCP was added according to the opposite volume gradient. By the same token, in the same way, mix fwYellow and gfasPurple, spisPink and amilCP, spisPink and gfasPurple.
Fig.2 experimental flow chart of colorimetriccard