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Induction of beta-galactosidase

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Introduction

Induction of beta-galactosidase Hypothesis: By adding lactose to the food source of Escherichia Coli, we will introduce the need for �-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 �-galactosidase into galactose and GNP, a yellow chemical. The presence of ONP will prove that �-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, �-galactosidase to be produced and the lactose to be digested into glucose and galactose. ...read more.

Middle

This enzyme catalyses the reaction that results in lactose being broken down into galactose and glucose. Lactose is a disaccharide, a type of sugar that is actually made up of two sugars - monosaccharides that are only made up of themselves. The two sugars that make up lactose are joined by a condensation reaction resulting in a glycosidic link. Here is a diagram of the breakdown of lactose; you can see the two monosaccharides glucose and galactose, and the glycosidic link: This reaction will not happen alone, it needs the enzyme beta-galactosidase to induce it. But, the bacteria E-coli does not have this enzyme operating normally in it's metabolic system, although it does have the genes to code for it; these are locked. The particular gene needed is prevented from being transcribed by RNA polymerase by a repressor molecule, which is coded for by a regulator gene that is always switched on. The repressor molecule (a type of protein) is usually bonded to the section of DNA which will result in the production of beta-galactose if coded for. If lactose is present, however, instead of somehow switching off the gene that produces the repressor molecule, as would be expected, it binds to the repressor molecule itself, drawing it away from the DNA and leaving it free to be transcribed. ...read more.

Conclusion

Therefore if I were to repeat the experiment I would ensure that we were very careful to prevent cross contamination by being much more meticulous in our chemical transfers and aseptic techniques. Test tube one was also somehow contaminated, this time with lactose - I can tell this because although it contained ONPG and e.coli, it should not have contained the lactose which began the pathway of chemical reactions that resulted in the breakdown of ONPG and the yellow colour. Therefore, although there is nothing wrong with method used, we were not careful enough in prevention of cross contamination and the results were skewed because of it. If we had been much more particular in our manner of carrying out the experiment, it would have given much more understandable and clear results. Conclusion: In conclusion, our results did match the hypothesis in that we managed to induce the production of beta-galasctosidase, and saw the resulting breakdown of ONPG and therefore also the breakdown of lactose into galactose and glucose, although we can only presume that this part of the experiment was successful as we had no way of measuring it. Unfortunately, some of our results were not as predicted because of the inprecise way we carried out our method, and two of the controls were distorted. ...read more.

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  1. Induction of beta-galactosidase

    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 �-galactosidase to test tube 4.

  2. Induction of Beta-galactosidase

    Hypothesis: By adding lactose to the culture of E. coli, this will introduce the need for Beta-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

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