Part:BBa_K2981014

Tyr promotes GFP expression in a tyrosine-sensitive measurement pathway

Tyr promotes GFP expression in a tyrosine-sensitive measurement pathway, which is part of our measurement of Tyr content.

Usage and Biology

Biosensor strains were grown in LB broth at 37 °C overnight. In a 96-well plate, The cells were diluted (1:50; v%) in 100μl of M9 (supplemented with 0.4% glucose, 0.24 mg/mL MgSO4, 11.1 μg/mL CaCl2, 0.3μM thiamine hydrochloride, and 50 μg/mL kanamycin) to each well, and 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 (485 nm/528 nm for GFP) and OD600 for each well every 30 minutes. Each group should be repeated for at least 3 times.
We wanted to determine if there was a dose-response relationship between the Phe concentration (concentration range 0-200μM) and the fold induction of the strain carrying ParoF-rfp. As shown in Fig. 1, it could be clearly observed that the increase in Tyr concentration had a significant repressive effect on ParoF (Fig. 1 A) and had a strong linear relationship (Fig. 1, R² = 0.9769).

Fig.1 (A) Tyr 24h fluorescence curve, (B) Linear regression curve of fluorescence intensity as a function of Tyr concentration at the 24th hour

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 M9 medium containing 1.8% urea and 0.05% uric acid. Because the Tyr content in the urine of PKU patients was around 20mM, we added the engineered bacteria to M9 medium (with 100μM Tyr) diluted 200 times urea and uric acid mother liquor, and set a blank control (no urea and urea added) and at 37°C, 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.
The results show that urea and uric acid inhibit cell growth and fluorescence induction(Fig.2).

Fig.1 fluorescence induction(A) and cell growth rate(B) were measured by adding the main components of urine (1.48mM urea and 14.8μM uric acid) to 100μM of Tyr

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1807
  Illegal BglII site found at 2149
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 710
  Illegal AgeI site found at 822
  Illegal AgeI site found at 1613
  
• 1000
  COMPATIBLE WITH RFC[1000]