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Induction of beta-galactosidase

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A3.13 - Induction of �-galactosidase The aim of this experiment is to demonstrate the induction of the lac operon in Escherichia coli (E. coli). The lac operon codes for the production of the protein �-galactosidase, which acts as an enzyme to break down lactose into glucose and galactose. �-galactosidase also breaks down a colourless synthetic compound called ONPG into the bright yellow ONP, which will allow us to see whether the enzyme is present. The lac operon is made up of 4 genes, each with a different role. If there is no lactose present, a repressor binds onto the operating region, preventing RNA polymerase from transcribing the �-galactosidase gene, so �-galactosidase is not produced. If lactose is present, it bonds with the repressor, removing it from the operating region, and so allowed the transcription of the �-galactosidase gene and the lactose permease gene (which allows lactose to be moved into the bacterial cell). Together, this means that �-galactosidase is only produced when it is needed to break down lactose in the bacterium's surroundings, conserving energy and resources. ...read more.


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: Factor Prevention Explanation How results will be affected Type of error Volume of bacteria culture, water and enzyme Use a pipette with small graduations More of the culture/water/enzyme may dilute the colour A greater volume may cause the colour change to not be visible Random, as the volume will be different Contamination of culture or equipment Sterilise equipment before use, use good microbiological techniques Unknown bacteria may contain �-galactosidase, causing the ONPG to break down Unknown bacteria may cause a colour change Random, as different bacteria may contaminate each mixture Volume of ONPG Use a pipette with small graduations More ONPG will require more �-galactosidase to break it down More ONPG may not undergo a colour change Random, as each mixture will have a different volume Dispersion of ONPG Shake ...read more.


culture near test tube Dilute with Virkon, then mop up with tissue and wash hands d Contamination of surroundings Place used pipette in disinfectant then autoclave EVACUATE AND RUSH EVERYONE TO HOSPITAL OR YOU WILL DIE e Burns from Bunsen burner Leave on safety flame when not in use Run under cold water 8 Methylbenzene is harmful and flammable Wear goggles, avoid skin contact and inhaling vapours, use a fume cupboard, keep away from Bunsen burner Rinse affected area, suffocate fire with heatproof mat/fire blanket 10 Spillage of bacteria mixture Ensure bung is tight See 4c Results Tube Contents Results 1 E. coli, no lactose Stayed colourless 2 E. coli, lactose Slowly turned pale yellow 3 Distilled water Stayed colourless 4 �-galactosidase Immediately turned bright yellow Conclusion From this, I can conclude that my prediction was right. The controls (tubes 3 and 4) show that it is the �-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 �-galactosidase. Tube 4 changed colour faster than tube 2 because the concentration of �-galactosidase is greater. Sophie Catt 28/1/08 ...read more.

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    3. Add �-galactosidase to test tube four. 4. Add 5 drops methylbenzene to each test tube, and 1cm� ONPG to each test tube. Bung all tubes and shake to disperse all chemicals. 5. Place all test tubes in a 35�C water bath and leave for at least 1 hour.

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