Difference between revisions of "Part:BBa K554010:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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This part was used to [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Device_2_testing:_SoxR.2FSoxS_system_regulating_GFP_production Device 2 testing: SoxR/SoxS system regulated GFP production]
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how you used this part and how it worked out.
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===Experimental results===
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In order to test the ability of Jedi Bacteria in sensing NO levels and activating genes linked to SoxS promoter, we built a Device testing, with GFP linked to SoxS promoter, as it is shown in the following schema (Figure 1):
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[[Image:UNICAMP_EMSE_GFP_SOX_device.jpg|center|700px]]
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<div align=center> '''Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_2:_NO_sensor.2FIL-10_producer Device 2] through replacement of IL-10 to GFP. '''</div>
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The ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and <font color=red>'''found to be FUNCTIONAL'''</font>. In the section below we present the detailed results.
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[[Image:UNICAMP_EMSE_GFP_SOX_device_result1.jpg|center|600px]]
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<div align=center>'''Figure 2: SoxS/SoxR fluorescence data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.'''</div>
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The NO is a product related to inflammatory response in vivo. To test the sensor ''in vitro'', we used [[http://en.wikipedia.org/wiki/Paraquat Paraquat]] as inducer, a molecule which imposes superoxide stress within the cell in a similar manner as nitric oxide.
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The experiment indicated that our sensor was capable of inducible production of GFP (or another protein controlled by the sensor). In addition, the protein induction can be modulated through varying the inducer concentration (as shown by Figure 2). Higher concentrations of Paraquat exhibited higher fluorescence levels, which indicates increased GFP concentrations.
  
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===Applications of BBa_K554010===
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The experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3. The [http://2009.igem.org/Team:Stanford/ProjectPage#Results_and_Analysis Stanford team (iGEM 2009)] showed that Paraquat can be toxic to E. coli cells, with growth inhibition at 60 – 80 µM.
  
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===User Reviews===
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[[Image:UNICAMP_EMSE_GFP_SOX_device_result2.jpg|center|600px]]
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<div align=center>'''Figure 3: SoxS/SoxR optical density data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.'''</div>
 
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Revision as of 15:09, 27 September 2011

This part was used to [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results#Device_2_testing:_SoxR.2FSoxS_system_regulating_GFP_production Device 2 testing: SoxR/SoxS system regulated GFP production]

Experimental results

In order to test the ability of Jedi Bacteria in sensing NO levels and activating genes linked to SoxS promoter, we built a Device testing, with GFP linked to SoxS promoter, as it is shown in the following schema (Figure 1):

UNICAMP EMSE GFP SOX device.jpg
Figure 1: Testing [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project#Device_2:_NO_sensor.2FIL-10_producer Device 2] through replacement of IL-10 to GFP.

The ability to recognize NO (nitric oxide), an inflammation signal molecule, was characterized for SoxR/SoxS sensor, and found to be FUNCTIONAL. In the section below we present the detailed results.

UNICAMP EMSE GFP SOX device result1.jpg
Figure 2: SoxS/SoxR fluorescence data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.

The NO is a product related to inflammatory response in vivo. To test the sensor in vitro, we used http://en.wikipedia.org/wiki/Paraquat Paraquat as inducer, a molecule which imposes superoxide stress within the cell in a similar manner as nitric oxide.

The experiment indicated that our sensor was capable of inducible production of GFP (or another protein controlled by the sensor). In addition, the protein induction can be modulated through varying the inducer concentration (as shown by Figure 2). Higher concentrations of Paraquat exhibited higher fluorescence levels, which indicates increased GFP concentrations.

The experimental concentrations of Paraquat did not show significant differences in cell growth as shown by OD levels in Figure 3. The [http://2009.igem.org/Team:Stanford/ProjectPage#Results_and_Analysis Stanford team (iGEM 2009)] showed that Paraquat can be toxic to E. coli cells, with growth inhibition at 60 – 80 µM.

UNICAMP EMSE GFP SOX device result2.jpg
Figure 3: SoxS/SoxR optical density data for concentrations 0, 5, 10, 20, 30 and 40 µM of inducer.

UNIQ16c45e8b5ed8cf7d-partinfo-00000000-QINU UNIQ16c45e8b5ed8cf7d-partinfo-00000001-QINU