Team:Calgary/Notebook/Protocols/decatecholization

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<p>This assay is used to verify that catechol 2,3-dioxygenase (XylE) is converting catechol to the yellow compound 2-hydroxymuconic semialdehyde (2-HMS). For this procedure we used to the newly constructed XylE part.  This part contains the TetR promoter the XylE gene and its native rbs site:</p>
<p>This assay is used to verify that catechol 2,3-dioxygenase (XylE) is converting catechol to the yellow compound 2-hydroxymuconic semialdehyde (2-HMS). For this procedure we used to the newly constructed XylE part.  This part contains the TetR promoter the XylE gene and its native rbs site:</p>
<ol>
<ol>
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<li>Grow up 2 ml overnight cultures in LB</li>
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<li>Grow up 2 ml <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">overnight cultures</a> in LB.</li>
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<li>Spin the cultures down and keep the supernatant </li>
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<li>Spin the cultures down and keep the supernatant.</li>
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<li>Bring the supernatant to a concentration of 0.1 M of Catechol by using a 1M catechol stock solution</li>
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<li>Bring the supernatant to a concentration of 0.1 M of Catechol by using a 1M catechol stock solution.</li>
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<li>The colour of the supernatant should change to  bright yellow very quickly ( about 30 seconds)s </li>
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<li>The colour of the supernatant should change to  bright yellow very quickly (in about 30 seconds).</li>
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<h2>GC-MS Catechol Assay</h2>
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In order to identify if the PetroBrick or <i>Micrococcus</i> was capable of further reducing catechol products, GC-MS was used. 
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<ol>
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<li>Grow overnight cultures of the PetroBrick in LB at 37<sup>o</sup>C and <i>xylE</i> construct, or <i>Microccus</i> in yeast tryptone broth at 26<sup>o</sup>C.
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<li>Subculture into the micrococcus or PetroBrick broth respectively and set up cultures with and without <i>xylE</i> as well as the appropriate control. 
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<li>Note: Prior to mixing, <i>xylE</i> and PetroBrick cultures were spun down at 4000 RPM for 5 min at room temperature and washed with 2x volume LB, spun down again, and then resuspended to the original volume to remove antibiotics which they were initially cultured in.
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<li>Add 10 mM catechol to each tube, mix well, and cover in tin foil. 
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<li>Culture the cells at the appropriate temperature for 72 hours.
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<li>Sonicate samples for 1 min using a Misonix Cell Disrupter set to Dial 11. 
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<li>Add 5 mL of ethyl acetate, shake well, and spin down at 4000 RPM for 5 min. at 4<sup>o</sup>C.
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<li> Add 1mL of the ethyl acetate to a tube and reduce the volume to 0.3mL through heating.
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<li> Samples were dried using sodium sulfate if any water layer was present and modified using trimethyl sylyl reagent.
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<li> Samples were ran onto the GC-MS (<a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/Carbazole_GC-MS_Analysis">see protocol</a>)
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Latest revision as of 02:54, 4 October 2012

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Catechol Assay in E. coli Cells

This assay is used to verify that catechol 2,3-dioxygenase (XylE) is converting catechol to the yellow compound 2-hydroxymuconic semialdehyde (2-HMS). For this procedure we used to the newly constructed XylE part. This part contains the TetR promoter the XylE gene and its native rbs site:

  1. Grow up 2 ml overnight cultures in LB.
  2. Spin the cultures down and keep the supernatant.
  3. Bring the supernatant to a concentration of 0.1 M of Catechol by using a 1M catechol stock solution.
  4. The colour of the supernatant should change to bright yellow very quickly (in about 30 seconds).

GC-MS Catechol Assay

In order to identify if the PetroBrick or Micrococcus was capable of further reducing catechol products, GC-MS was used.
  1. Grow overnight cultures of the PetroBrick in LB at 37oC and xylE construct, or Microccus in yeast tryptone broth at 26oC.
  2. Subculture into the micrococcus or PetroBrick broth respectively and set up cultures with and without xylE as well as the appropriate control.
  3. Note: Prior to mixing, xylE and PetroBrick cultures were spun down at 4000 RPM for 5 min at room temperature and washed with 2x volume LB, spun down again, and then resuspended to the original volume to remove antibiotics which they were initially cultured in.
  4. Add 10 mM catechol to each tube, mix well, and cover in tin foil.
  5. Culture the cells at the appropriate temperature for 72 hours.
  6. Sonicate samples for 1 min using a Misonix Cell Disrupter set to Dial 11.
  7. Add 5 mL of ethyl acetate, shake well, and spin down at 4000 RPM for 5 min. at 4oC.
  8. Add 1mL of the ethyl acetate to a tube and reduce the volume to 0.3mL through heating.
  9. Samples were dried using sodium sulfate if any water layer was present and modified using trimethyl sylyl reagent.
  10. Samples were ran onto the GC-MS (see protocol)








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