Part:BBa_K649401:Experience

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Applications of BBa_K649401

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 is MG1655.

Plasmids transformed into E.coli in this study are shown in TABLE 1.

TABLE 1. Expression plasmids used for Charcterization of rocF and Arg box

designation	pSB3K3	pSB1C3
mock	PlacIQ	Alcohol-dehydrogenase(promoter-less)
rocF	Ptrc-rocF	Alcohol-dehydrogenase(promoter-less)
rocF + Arg box	Ptrc-rocF	Arg box

Strain MG1655 was transformed with either mock, rocF or rocF + Arg box. As shown in Table 1, rocF gene was introduced on pSB3K3 (BBa_K649301) and Arg box(BBa_K649401) was introduced on pSB1C3 respectively.

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



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

[Discussion]
As a way to derepress arginine biosynthesis, we introduced Arg box on our part BBa_K649401 in addition to rocF gene on our part BBa_K649301. In this case, more urea was produced compared to that when only rocF gene was introduced. These results show that Arg boxes are effectively derepressing arginine production by deactivating the arginine repressor.

[Improvement on this biobrick part]
Team UT-Tokyo 2012 [http://2012.igem.org/Team:UT-Tokyo/Project/Inhibition/Discussion] improved this biobrick part creating new, stronger part Part:BBa_K778003 Part:BBa_K778006.

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