Difference between revisions of "Part:BBa K649402:Experience"

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Applications of BBa_K649402

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. JE6852

Plasmid transformed into E.coli in this study

1. mock
2. Ptrc-rbs-rocF(BBa_K649301)
3. Ptrc-rbs-rocF-Arg Box(BBa_K649402)

[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 ampicillin 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. 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
   
   ODSAMPLE, ODBLANK and ODSTANDARD are OD450 values of sample, standard and blank, respectively.

[Discussion]

In MG1655(argR+) and JD24293(argR-), addition of Arg box sequence led to little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that pSB6A1 is a low-copy-number plasmid, in contrast to high-copy number used in the previous report. A low-copy-number plasmid is not capable of introducing enough number of Arg boxes to effectively deactivate the arginine repressor. Both of the plasmids containing rocF gene in the stain JD24293(argR -) produce urea more efficiently than those in MG1655.

These results are in line with the fact that JD24293 carries argR (a gene which codes arginine repressor) loss-of-function mutant, which means deactivation of arginine repressor by Arg boxes is not needed and addition of the Arg box does not result in a significant increase of urea production.

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