Urea-cooler Assay data

1. Characterization of rocF and Arg box

1.1 Materials

Expression plasmids used in this study are shown in Table 1.

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

designation	pSB3K3	pSB6A1
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, tocF or rocF + Arg. As shown in Table 1, rocF gene was introduced on pSB3K3 and Arg boxes were introduced on pSB6A1.

1.2 Methods

1.2.1 Preparation of samples for urea concentration assay

1. A single colony of cells transformed with engineered plasmids (mock,rocF or rocF+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics 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.

1.2.2 Urea concentration assay

Urea concentrations of the samples were determined colorimetrically with DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems.
Detailed methods are as follows.

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 munites 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.

Standard curve for coloring reaction in urea assay

To generate standard curve, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate in the same way as the samples. Standard curve is shown in Fig.1.

Fig.1 Standard curve for coloring reaction in urea assay

1.3 Results

Each sample was assayed in duplicate urea concentration detected in each sample is shown in Table 2. Fig. 2 shows the average of these 2 values.

TABLE 2. Urea concentrations detected in duplicated

colony No.	mock	rocF	rocF+Arg box
#1	1.9	4.9	7.3
#2	0.75	4.3	7.2
Average	1.3	4.6	7.2
B.D.	0.80	0.44	0.080

Fig.2 The average of concentration values detected in duplicate

2. Characterization of Ptrc-RBS-rocF-Argbox

2.1 Materials

Expression plasmids used in this study are listed in table 1.

TABLE 3. Expression plasmids used in this study

designation	Parent vector	Introduced sequence(s)
Mock (3K3)	pSB3k3	PlacIQ
rocF (3K3)	pSB3K3	Ptrc-rocF
rocF+Arg box (3K3)	pSB3K3	Ptrc-rocF-Arg box
Mock (6A1)	pSB6A1	gfp (promoter-less)
rocF (6A1)	pSB6A1	Ptrc-rocF
rocF+Arg box (6A1)	pSB6A1	Ptrc-rocF-Arg box

In one experiment, MG1655 (argR +) and JE6852 (argR -) were respectively transformed with either mock(3K3), rocF(3K3) or rocF-Arg box(3K3). In another experiment, MG1655 (argR +) and JD24293 (argR -) were respectively transformed with either mock(6A1), rocF(6A1) or rocF-Arg box(6A1).

2.2 Methods

2.2.1 Preparation of samples for urea concentration assay

1. A single colony of cells transformed with engineered plasmids (mock,rocF or rocF+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics 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.

2.2.2 Urea concentration assay

Urea concentrations of the samples were determined colorimetrically with DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems.
Detailed methods are as follows.

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 munites 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.

Standard curve for coloring reaction in urea assay

To generate standard curve, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate in the same way as the samples. Standard curve is shown in Fig.1.

Fig.3 Standard curve for coloring reaction in urea assay

2.3 Results

Fig.4 Urea concentration detected in bacterial samples on pSB3K3

Fig.4 Urea concentration detected in bacterial samples on pSB3K3