Difference between revisions of "Part:BBa K2981013"

Revision as of 13:28, 19 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, 0.3 μM 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).

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

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

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 ===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, 0.3 μM 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.
+<br>
+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).
 [[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]]