Team:Tokyo Tech/Projects/Urea-cooler/data
From 2011.igem.org
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.
designation | pSB3K3 | pSB1C3 |
mock | PlacIQ | Alcohol-dehydrogenase(promoter-less) |
rocF | Ptrc-rocF | Alcohol-dehydrogenase(promoter-less) |
rocF+Arg box | Ptrc-rocF | Arg box |
1.2 Methods
1.2.1 Preparation of samples for urea concentration assay
- 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℃.
- The saturated culture was diluted 50-fold, grown till the log phase (OD600 = 0.5).
- The culture was induced with 1 mM IPTG at 37℃ for 1 hour.
- 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.
- 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.
- 200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.
- The mixture was incubated for 20 munites at room temperature.
-
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.
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 |
![](https://static.igem.org/mediawiki/2011/d/de/Characterization_rocF_and_Arg_box.png)
2. Characterization of Ptrc-RBS-rocF-Argbox
2.1 Materials
Expression plasmids used in this study are listed in table 1.
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
- 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℃.
- The saturated culture was diluted 50-fold, grown till the log phase (OD600 = 0.5).
- The culture was induced with 1 mM IPTG at 37℃ for 1 hour.
- 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.
- 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.
- 200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.
- The mixture was incubated for 20 munites at room temperature.
-
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
![](https://static.igem.org/mediawiki/2011/9/90/RocF-Arg_box_on_pSB3K3.png)
![](https://static.igem.org/mediawiki/2011/5/5b/RocF-Arg_box_on_pSB6A1.png)
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.