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Part:BBa_K649301:Experience

Designed by: Goshi Sugano   Group: iGEM11_Tokyo_Tech   (2011-09-19)

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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 was done by Natsuki Kubo.

[Sample]
E.coli strains used in this study

    MG1655

Plasmid transformed into E.coli in this study

    Designation Parental vector Introduced sequence(s) Part No.
    mock pSB3K3 PlacIQ -
    rocF pSB3K3 Ptrc-rocF BBa_K649301


[Method]
Preparation of samples for urea concentration assay

  1. A single colony of cells transformed with engineered plasmids (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. 1.5 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. The mixture was incubated for 20 minutes at room temperature.
  4. Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as
    Kit_equation.png
    ODSAMPLE, ODBLANK and ODSTANDARD are OD450 values of sample, standard and blank, respectively.

    5. Each sample was assayed in triplicate and the averatge of three values was calculataed.

[Discussion]
In MG1655(argR +), addition of Trc promoter-rocF led to production of more urea compared to the bare backbone pSB3K3 as expected. These results show that insertion of rocF resulted in arginase production, therefore completing the urea cycle in E.coli.

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