Difference between revisions of "Part:BBa K649401:Experience"
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===Applications of BBa_K649401=== | ===Applications of BBa_K649401=== | ||
− | [[Image: | + | [[Image:Arg_box_on_1C3.png|thumb|right|500px|Urea concentration in growth media 1 hour after IPTG induction.<br /> |
− | This work | + | This work was done by Natsuki Kubo.]] |
'''[Sample]'''<br /> | '''[Sample]'''<br /> | ||
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<th>designation</th> | <th>designation</th> | ||
<th>pSB3K3</th> | <th>pSB3K3</th> | ||
− | <th> | + | <th>pSB1C3</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
</table><br /> | </table><br /> | ||
− | Strain MG1655 was transformed with either mock, <i>rocF</i> or <i>rocF</i> + Arg box. As shown in Table 1, <i>rocF</i> gene was introduced on pSB3K3 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K649301 BBa_K649301]) and Arg box([https://parts.igem.org/wiki/index.php?title=Part:BBa_K649401 BBa_K649401]) was introduced on | + | Strain MG1655 was transformed with either mock, <i>rocF</i> or <i>rocF</i> + Arg box. As shown in Table 1, <i>rocF</i> gene was introduced on pSB3K3 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K649301 BBa_K649301]) and Arg box([https://parts.igem.org/wiki/index.php?title=Part:BBa_K649401 BBa_K649401]) was introduced on pSB1C3 respectively. <br /> |
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<ol> | <ol> | ||
<li> | <li> | ||
− | 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℃ | + | 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 chloramphenicol and grown to saturation at 37℃. |
</li> | </li> | ||
<li> | <li> | ||
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'''[Discussion]'''<br /> | '''[Discussion]'''<br /> | ||
− | As a way to derepress arginine biosynthesis we introduced Arg box on our part BBa_K649401 in addition to <i>rocF</i> gene on our part [https://parts.igem.org/Part:BBa_K649301 BBa_K649301]. In this case, more urea was produced compared that when only <i>rocF</i> gene | + | As a way to derepress arginine biosynthesis, we introduced Arg box on our part BBa_K649401 in addition to <i>rocF</i> gene on our part [https://parts.igem.org/Part:BBa_K649301 BBa_K649301]. In this case, more urea was produced compared to that when only <i>rocF</i> gene was introduced. These results show that Arg boxes are effectively derepressing arginine production by deactivating the arginine repressor. |
+ | |||
+ | '''[Improvement on this biobrick part]'''<br /> | ||
+ | Team UT-Tokyo 2012 [http://2012.igem.org/Team:UT-Tokyo/Project/Inhibition/Discussion] improved this biobrick part | ||
+ | creating new, stronger part [[Part:BBa_K778003]] [[Part:BBa_K778006]]. | ||
===User Reviews=== | ===User Reviews=== |
Latest revision as of 06:10, 29 September 2012
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_K649401
[Sample]
E.coli strains used in this study is MG1655.
Plasmids transformed into E.coli 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 |
Strain MG1655 was transformed with either mock, rocF or rocF + Arg box. As shown in Table 1, rocF gene was introduced on pSB3K3 (BBa_K649301) and Arg box(BBa_K649401) was introduced on pSB1C3 respectively.
[Method]
Preparation of samples for urea concentration assay
- 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 chloramphenicol 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.
Urea concentration assay
- 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 minutes 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.
Each sample was assayed in triplicate and the averatge of three values were calculataed.
[Discussion]
As a way to derepress arginine biosynthesis, we introduced Arg box on our part BBa_K649401 in addition to rocF gene on our part BBa_K649301. In this case, more urea was produced compared to that when only rocF gene was introduced. These results show that Arg boxes are effectively derepressing arginine production by deactivating the arginine repressor.
[Improvement on this biobrick part]
Team UT-Tokyo 2012 [http://2012.igem.org/Team:UT-Tokyo/Project/Inhibition/Discussion] improved this biobrick part
creating new, stronger part Part:BBa_K778003 Part:BBa_K778006.
User Reviews
UNIQ710d29f2fa6b4735-partinfo-00000000-QINU UNIQ710d29f2fa6b4735-partinfo-00000001-QINU