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Part:BBa_K649402:Experience

Designed by: Maiko Hayashi   Group: iGEM11_Tokyo_Tech   (2011-09-19)

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Applications of BBa_K649402

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

  1. MG1655
  2. JE6852

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
    rocF-Arg box pSB3K3 Ptrc-rocF-Arg box BBa_K649402

    [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 were calculataed.

    [Discussion]
    In MG1655(argR+) and JE6852(argR-), introduction of rocF gene lead to production of more urea. However, introduction of Arg boxes in addition to rocF gene showed little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that pSB3K3 is a low-copy-number plasmid. We assume that a low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. In contrtact, when Arg boxes were intoduced on a high-copy-number plasmid (pSB1C3) using our part BBa_K649401 separetely with rocF gene, Arg boxes worked effectively.
    Both of the plasmids containing rocF gene in the stain JE6852(argR -) produce urea more efficiently than those in MG1655(argR+).

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