Difference between revisions of "Part:BBa K649301:Experience"

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Applications of BBa_K649301

Because arginase is constitutively expressed, the expression level of urea in E. coli transformed with BBa_K649301 was higher than mock E. coli.

Urea concentration in growth media 1 hour after IPTG induction.
This work is done by Natsuki Kubo.

[Sample]
E.coli strains used in this study

1. MG1655
2. JD24293

Plasmid transformed into E.coli in this study

1. mock
2. Ptrc-rbs-rocF
3. Ptrc-rbs-rocF-Arg Box

[Method]
Preparation of samples for urea concentration assay

1. A single colony of cells transformed with mock, Ptrc-rocF or Ptrc-rocF-Arg box was inoculated into 3 mL of LB with kanamycin and grown to saturation at 37℃
2. The saturated culture was diluted 50-fold, grown till the log phase (OD₆₀₀ = 0.5).
3. The culture was induced with 1 mM IPTG at 37 ℃ for 1 hour.
4. 2 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.

Urea concentration assay

1. 10 µL of the supernatant fluid from each sample, 10 µL blank(LB),and 10 µL standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.
2. 200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.
3. Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as
   [[Image:Kit_equation.png|left|200px]

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