Assay Method and Results

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	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. As shown in Table 1, rocF gene was introduced on pSB3K3 and Arg boxes were introduced on pSB1C3.

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°C.
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°C 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.

Linear plot of OD₄₅₀ vs. urea concentraion

To test linearlity between OD₄₅₀ vs. urea concentraion, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate with this urea assay kit in the same way as our bacterial samples. Plot of OD₄₅₀ values vs. urea concentraions we tested showed very good linearlity as shown in Fig.1.

fig.1 Linear plot of OD₄₅₀ vs. urea concentraion

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°C.
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°C 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.5 Urea concentration detected in bacterial samples on pSB6A1

In MG1655(argR +), addition of Trc promoter-rocF led to more production of urea compared to the bare backbone pSB6A1 as expected. These results show that insertion of rocF resulted in arginase production as expected, therefore completing the urea cycle in E. coli. In the same strain, however, 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. In both cases when pSB3K3 was used and when pSB6A1 was used, similer results were obtained.