ONPG is also broken down by β-galactosidase, it produces ONP a visible yellow liquid and galactose.
If yellow is seen, active β-galactosidase is present.
Equipment/Setup
Method
- Wipe down all work surfaces with disinfectant.
- Label four test tubes 1 to 4.
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Add 5cm3 sodium phosphate solution to each test tube.
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Using aseptic techniques (sterilising the test tube before each transfer using a Bunsen burner and a new sterilised pipette), transfer 1cm3 of the E coli bred without lactose into test tube 1.
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Using the same technique, transfer 1cm3 of the E coli bred with lactose into test tube 2.
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Next, add 1cm3 of distilled water to test tube 3. This is the negative control.
- Add the enzyme β-galactosidase into test tube 4.
- Now using a pipette, add five drops of methylbenzene to each test tube.
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Add 1cm3 of the ONPG solution to each test tube.
- Cover each test tube with a stretched peace of parafilm, shake well to dispense the ONPG.
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Place all four test tubes into a water bath set at 35oC.
- After leaving for a set period of time, remove test tubes and compare developed colours.
- Record observations.
Safety
All enzymes should be treated as allergens; therefore avoid contact and inhalation. Do not swallow.
Methylbenzene is flammable and harmful if inhaled. Keep away from flames. The vapour causes irritation to the eyes and evaporation should only take place in a fume cupboard. Avoid contact with skin and eyes; eye protection must be worn.
Aseptic techniques must be used to avoid any contamination due to spillage of micro-organisms.
Any spills should be dealt with immediately and correctly.
No eating or drinking anywhere near the experiment.
Cover all skin cuts with a waterproof plaster. Be sure to wipe all surfaces down with disinfectant and wash hands with soap and water before the practical.
Aseptic techniques
All equipment should be sterile before usage. (Autoclave glassware; using a low Bunsen flame to decontaminate glass, metal wire before the transfer of bacteria and wash plastic with 70% ethanol.)
This is to avoid cross-contamination between equipment.
Make sure to place all used equipment straight into disinfectant after use.
After I have done this experiment I will compare the colour of the solution in the test tubes. A yellow colour will show the presence of the enzyme β-galactosidase. This should be produced in test tube 2, containing the E coli bred in a lactose broth and test tube 4, containing the β-galactosidase (the positive control)
Results
I think that these results are valid as they are related to my hypothesis. Not only are my results related, they prove my hypothesis correct.
β-Galactosidase was induced in the test tube containing the bacteria grown in lactose as it turned yellow along side the test tube containing β-galactosidase.
The results are also reliable as the experiment was repeated eight times, with the same results achieved every time. These results are both accurate and precise as they all show the same results, which are in direct correspondence with the truth according to biological facts.
There were a few errors related to this experiment. A random error could be related to the fact that there may have been different shades of yellow produced. This could be related to human error and overshooting measurements on the pipettes.
As the results of this experiment were completely judged by eye, there were not any qualitative results, only quantitative. But this would really be more relevant if I was testing for the amount of β-Galactosidase that was induced. (But that’s another experiment! ☺)
Conclusion
My results conclude that β-galactosidase is induced from E-coli grown in lactose. This is because genes are expressed in cells when the coded protein products are needed.
If lactose is in the E-coli growth medium it must be broken down before it can be used in respiration. This bacteria contains encoded in it a gene for the enzyme β-galactosidase which breaks down lactose. The presence of lactose activates this gene so the enzyme is then produce.
This proves that genes can be “switched on and off” (called de-repression).
The lack of lactose will therefore deactivate the gene co the enzyme is not produced.
These results are reliable. The experiment was repeated eight times each giving the same results. The results were done by eye, quantitatively rather than qualitatively, so this could be classified as a limitation.
This could be overcome by using a colorimeter to generate qualitative results to clarify definite values.
In summary, I planned an investigation to find out whether β-Galactosidase will only be induced in the presence of lactose. β-Galactosidase is the enzyme that breaks down lactose into glucose and galactose.
To find this out I used two types of E coli, one bred in the presence of lactose and one bred without lactose.
My results concluded that the β-Galactosidase was only induced with lactose present. (The solution turned yellow)
This, in conclusion, proved to me that genes can be switched on and off by way of de-repression.