Difference between revisions of "Part:BBa K649402:Experience"
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each sample was assayed in triplicate and the averatge of three values were calculataed.<br />
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Each sample was assayed in triplicate and the averatge of three values were calculataed.<br />
 
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'''[Discussion]'''<br>
 
'''[Discussion]'''<br>
 
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In MG1655(argR+) and JD24293(argR-), 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.
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In MG1655(argR+) and JD24293(argR-), introduction of <i>rocF<i/> gene lead to production of more urea. However, introduction of Arg boxes in addition to <i>rocF<i/> gene showed little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that pSB3K3 is a low-copy-number plasmid. We assume that 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 JE6852(argR -) produce urea more efficiently than those in MG1655(argR+).
 
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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.
 
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===User Reviews===
 
===User Reviews===

Revision as of 17:32, 26 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_K649402

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. JE6852

Plasmid transformed into E.coli in this study

  1. mock
  2. Ptrc-rbs-rocF(BBa_K649301)
  3. Ptrc-rbs-rocF-Arg Box(BBa_K649402)

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

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

[Discussion]

In MG1655(argR+) and JD24293(argR-), introduction of rocF<i/> gene lead to production of more urea. However, introduction of Arg boxes in addition to <i>rocF<i/> gene showed little change in urea production. The reason why the effect of Arg boxes was not apparent is probably that pSB3K3 is a low-copy-number plasmid. We assume that 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 JE6852(argR -) produce urea more efficiently than those in MG1655(argR+). </p>

User Reviews

UNIQ52ac503a76fe6dd1-partinfo-00000000-QINU UNIQ52ac503a76fe6dd1-partinfo-00000001-QINU