Induction of beta-galactosidase

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Induction of beta-galactosidase

Hypothesis:

By adding lactose to the food source of Escherichia Coli, we will introduce the need for ß-galactosidase in the bacteria. The presence of lactose will overcome the forces holding the repressor molecule to the gene which will code for the amino acids which will form the enzyme, therefore the RNA polymerase will be able to bind to the gene and cause the DNA to unwind, allowing mRNA to be produced that codes for the genes.

Once the enzyme is produced, it will break down the disaccharide lactose into the monosaccharides glucose and galactose, which are then used in respiration.

We will add ONPG to the mixture, which is also catalysed by ß-galactosidase into galactose and GNP, a yellow chemical. The presence of ONP will prove that ß-galactosidase is present in the mixture.

Therefore upon adding lactose to a mixture of bacteria and ONPG, ONP will be produced.

Aim:

We will attempt to find out whether adding lactose in with bacteria will cause the genes which code for beta-galactosidase to be unlocked, ß-galactosidase to be produced and the lactose to be digested into glucose and galactose.

Safety Precautions:

  • Wear gloves and lab coat at all times.
  • Ensure area used is thoroughly sterilised and there are no stray chairs or bags on the floor and the desk is entirely clear.
  • Ensure all equipment to be used is sterile.
  • Keep a pot of sterilising solution close by, and place all equipment in there once used.
  • Always be careful to avoid cross contamination of chemicals and mixtures.
  • Wash hands before and after experiment.
  • Always use aseptic techniques to transfer chemicals.
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Method:

  1. Add 5cm³ sodium phosphate to four test tubes.
  2. Transfer 1cm³ E. coli in nutrient broth to the first test tube, and 1cm³ E. coli plus nutrient broth (lactose) to the third.
  3. Add ß-galactosidase to test tube four.
  4. Add 5 drops methylbenzene to each test tube, and 1cm³ 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.
  6. Place a sample from each test tube into a colourimeter and record results.

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