| Addition of catechol to a a culture of cells constitutively expressing C2,30 enzyme results into production of yellow colonies on plate or yellow liquid cultures if E.coli is grown in liquid medium. In a lab where a spectrophotometer is readily available anyone can accurately quantify production of even the smallest quantity of the yellow 2-hydroxymuconic semialdehyde product. However, as our biological detector is intended for field use where sophisticated spectroscopic equipment is not available, it was obvious that we needed to establish the threshold value at which a positive result (yellow color) would be detectable by the naked eye.
The set up of this experiment involved preparation of XylE expressing E.coli cultures at an O.D. of 0.5 in LB and M9 medium. Then a gradient of catechol concentrations was prepared and added to the cell cultures. A group of 5 individuals, but not the lab member preparing the test samples, were given the test cultures and asked where on the gradient they were not able anymore to see the yellow color. The 2 concentrations where the transition from yellow to colorless occurred were identified and a second gradient of catechol concentrations was prepared. In this second gradient the gap between the 2 concentration was analysed. The highest catechol concentration was the least yellow from the first part of the experiment, while the lowest catechol concentration was the first "colourless" concentration from the first part of the experiment. Again the group of 5 testers identified the transition concentrations from yellow to colorless.
|
|
| Threshold value experiment was conducted on a 96 well plates. Yellow wells are the positive tests, LB color and M9 color mediums wells are the negative tests.
|
|
"
