Induction of beta-galactosidase

Prediction

I predict that the bacteria grown in lactose will have ß-galactosidase present, so the ONPG will be broken down and the mixture will turn yellow due to ONP. The bacteria grown without lactose will have no need for ß-galactosidase, so the ONPG will not be broken down and so will remain colourless. I will also use 2 controls: a negative control of distilled water (which will remain colourless, to show that the ONPG does not break down on its own) and a positive control of ß-galactosidase (which will turn yellow, to show that this is what breaks ONPG down).

Method

1. Wipe down the bench with disinfectant.
2. Label 4 test tubes 1-4.
3. Add 5cm3 sodium phosphate solution to each test tube.
4. Using aseptic techniques, transfer 1cm3 of the E. coli in nutrient broth without lactose to test tube 1.
5. Using aseptic techniques, transfer 1cm3 of the E. coli in nutrient broth with lactose to test tube 2.
6. Using the same techniques, add 1cm3 of distilled water to test tube 3.
7. Add 1cm3 ß-galactosidase to test tube 4.
8. Add 5 drops of methylbenzene to each test tube (in a fume cupboard).
9. Add 1cm3 ONPG solution to each test tube.
10. Fit a bung to each test tube and shake.
11. Put all the test tubes in a water bath set at 35°C.
12. Compare the colours as they develop.

Variables

The independent variable is whether or not the E. coli has been growing in lactose, and the dependant variable is whether or not the mixture turns yellow due to ONP.

I will be controlling the following variables:

Safety and Ethical Issues

There are no ethical issues involved, as bacteria are being used instead of animals.

Results

Conclusion

From this, I can conclude that my prediction was right. The controls (tubes 3 and 4) show that it is the ß-galactosidase that changes the colour in the test tube, and the fact that test tube 2 changed colour shows that it is the presence of lactose which induces the production of ß-galactosidase. Tube 4 changed colour faster than tube 2 because the concentration of ß-galactosidase is greater.