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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 37^oC and xylE construct, or Microccus in yeast tryptone broth at 26^oC.
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 and collect any products produced using the extraction protocol page.