Difference between revisions of "Part:BBa K649301:Experience"

(→‎Applications of BBa_K649301)
(→‎Applications of BBa_K649301)
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Because arginase is constitutively expressed, the expression level of urea in E. coli transformed with BBa_K649301 was higher than mock E. coli.  
 
Because arginase is constitutively expressed, the expression level of urea in E. coli transformed with BBa_K649301 was higher than mock E. coli.  
  
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[[Image:BBa K649001 graph3.png|thumb|right|500px|Effect of 3OC12-HSL induction on fluorescence intensity<br>This work is done by Takuya Tsubaki.]]
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[[Image:BBa K649301 graph(2).png|thumb|center|500px|Urea concentration in growth media 1 hour after IPTG induction.<br />
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This work is done by Natsuki Kubo.]]
 +
 
 +
'''[Sample]'''
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<i> E.coli</i> strains used in this study
 +
<ol>
 +
<li>MG1655</li>
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<li>JD24293</li>
 +
</ol>
 +
 
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Plasmid transformed into <i> E.coli</i> in this study
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<ol>
 +
<li>mock</li>
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<li>Ptrc-rbs-rocF</li>
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<li>Ptrc-rbs-rocF-Arg Box</li>
 +
</ol>
 +
 
 +
'''[Method]'''
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Preparation of samples for urea concentration assay
 +
<ol>
 +
<li>
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A single colony of cells transformed with mock, Ptrc-rocF or Ptrc-rocF-Arg box was inoculated into 3 mL of LB with kanamycin and grown to saturation at 37℃
 +
</li>
 +
<li>
 +
The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).
 +
</li>
 +
<li>
 +
The culture was induced with 1 mM IPTG at 37 ℃ for 1 hour.
 +
</li>
 +
<li>
 +
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.
 +
</li>
 +
</ol>
 +
Urea concentration assay
 +
<ol>
 +
<li>
 +
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.
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</li>
 +
<li>
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200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.
 +
</li>
 +
<li>
 +
Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br>
 +
[[Image:Kit_equation.png|left|200px]
 +
</li>
 +
</ol>
  
 
===User Reviews===
 
===User Reviews===

Revision as of 20:19, 5 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K649301

Because arginase is constitutively expressed, the expression level of urea in E. coli transformed with BBa_K649301 was higher than mock E. coli.

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. JD24293

Plasmid transformed into E.coli in this study

  1. mock
  2. Ptrc-rbs-rocF
  3. Ptrc-rbs-rocF-Arg Box

[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 kanamycin 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. Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as
    [[Image:Kit_equation.png|left|200px]

User Reviews

UNIQ2e12a91f72892745-partinfo-00000000-QINU UNIQ2e12a91f72892745-partinfo-00000001-QINU