Introduce the background theory: (a) What is the biochemical function of β-galactosidase in the bacterial cell? (b) Explain how the expression of the lac operon (which includes the lacZ gene) is regulated. Show a diagram of the operon, including all regulatory DNA sections, chemicals and proteins/enzymes, to help with your explanation. (c) What is IPTG? (d) What is chloramphenicol, how does it work, and what is its purpose here? (e) How is β-galactosidase activity being measured here? Aims (1 Mark) Write an aim for the experiment, for example: ‘To assess the effects of inducers (lactose, IPTG) and repressors (glucose) on the expression of lacZ gene’. Materials and Methods (4 Marks)Keep it concise (10-12 lines). Use third person speech, past tense. No dot points. Do not copy the prac info word for word. All important details must be present, including concentrations of IPTG, chloramphenicol, ONPG, CTAB, components of the growth media, method of preparation of the bacterial cell suspension that was tested. Discussion (12 Marks) Discuss all important points such as: (a) State the molar absorption data you determined for the standard curve. Include comments on any inconsistencies or irregularities in it. (It is fine if you didn’t have any! Say so!) Comment and explain your actual results. (3 marks) (b) Summarise and discuss the trends of values you obtained of TEA for various conditions: lactose, IPTG, Glucose, Glycerol and chloramphenicol. What was the effect of the added substance on the β-galactosidase activity? (3 marks) (c) What did you expect in each of these flasks? Did the results agree with your expectations? Explain the biochemical principles behind your results. (4 marks) (d) Comment on the potential applications of this enzyme. (2 marks) Conclusions (1 Mark) Keep this concise (not more than 4-5 statements). Give the conclusions (identify inducers / repressors, state TEA of the inducers/repressors, etc.) References (2 Marks) Use a few relevant references in the introduction and discussion sections, and also methods if you wish to. Use reputable sources such as textbooks and journal articles. Wikipedia is NOT a reputable source. Note the formatting styles for referencing. References should be cited in the introduction and discussion sections if relevant. This experiment we have done in the class The broad aims of this practical exercise is to investigate the effects of certain factors (such as glucose, galactose, glycerol, lactose, etc.) in the cell culture medium on the induction of expression of β-galactosidase (β-gal). Introduction β-gal is an enzyme that hydrolyses the β-bond of the disaccharide sugar lactose to break it down to its constituent monomer sugars, glucose and galactose. Escherichia coli can grow on lactose by producing this enzyme, which hydrolyses lactose; the E. coli cells then use the sugars for cell growth. Some strains produce the enzyme constitutively but there is a considerable advantage to be gained by the organism having an inducible β-gal, where the expression of the β-galactosidase gene (lacZ) that encodes this enzyme is induced by the substrate (refer to your textbook for details of lac operon and its control). However, lactose is not the only inducer of the enzyme, nor is it the most effective inducer. There is a series of synthetic phenyl β-D-thiogalactosides that are more effective than lactose. The most effective of these is isopropyl-β-D-thiogalactoside (IPTG). In biotechnology work, this enzyme (and other inducible enzymes) is often used for various applications, for example: ï‚· As a reporter gene, to detect the activity of a ‘promoter’ i.e., section of DNA that regulates the expression of a gene. If the appropriate promoter has been cloned next to the gene that encodes this enzyme, the enzyme will be expressed and its activity can be assayed. ï‚· To test the success of a cloning experiment involving cloning of a foreign DNA into a plasmid or other cloning vector. If the cloned DNA fragment disrupts this gene on the plasmid, the enzyme is not made.