Difference between revisions of "Part:BBa K2981013"
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===Usage and Biology===
 
===Usage and Biology===
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[[File:T--NWU-China--result-Phe.jpg|900px|thumb|none|alt=Phe 24h fluorescence curve.|Figure 1. Phe 24h fluorescence curve]]
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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 Phe 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.
 
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[[File:T--NWU-China--result-Phe24h.jpg|900px|thumb|none|alt=Phe 24th hour fluorescence curve.|Figure 1. Phe 24th hour linear analysis of fluorescence curve]]
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We wanted to determine if there is a dose-response relationship between the Phe concentration (concentration range 0-200μM) and the fold induction of the strain carrying PtyrP-rfp. As shown in Fig. 1A, it could be clearly observed that the increase in Phe concentration had a significant effect on the activation of PtyrP and was linear (Fig. 1B, R² =0.9873).
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[[File:T--NWU-China--Layout-Phe.jpg|900px|thumb|none|alt=Phe 24h fluorescence curve.|Fig.1 (A) Phe 24h fluorescence curve, (B) Linear regression curve of fluorescence intensity as a function of Phe concentration at the 24th hour]]
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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 Phe content in the urine of PKU patients was around 20 mM (H.A. Harper et al., 1973), we added the engineered bacteria to M9 medium (with 100μM Phe) 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.
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The results show that urea and uric acid inhibit cell growth and fluorescence induction(Fig.2).
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[[File:T--NWU-China--Urea-Phe.jpg|900px|thumb|none|alt=Phe 24h fluorescence curve.|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 Phe]]
  
 
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Latest revision as of 01:58, 22 October 2019


For phenylalanine-sensitive measurement pathways

For phenylalanine-sensitive measurement pathways, Phe promotes GFP expression, which is part of our measurement of Phe 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 Phe 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 is a dose-response relationship between the Phe concentration (concentration range 0-200μM) and the fold induction of the strain carrying PtyrP-rfp. As shown in Fig. 1A, it could be clearly observed that the increase in Phe concentration had a significant effect on the activation of PtyrP and was linear (Fig. 1B, R² =0.9873).

Phe 24h fluorescence curve.
Fig.1 (A) Phe 24h fluorescence curve, (B) Linear regression curve of fluorescence intensity as a function of Phe 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 Phe content in the urine of PKU patients was around 20 mM (H.A. Harper et al., 1973), we added the engineered bacteria to M9 medium (with 100μM Phe) 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).

Phe 24h fluorescence curve.
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 Phe

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 1822
    Illegal BglII site found at 2164
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1628
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 800