21.4 Recombinant DNA technology Calculation worksheet. AQA Biology. Calculating the efficiency of DNA transfer during genetic engineering

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1 Calculating the efficiency of DNA transfer during genetic engineering Specification references MS 0.1, MS 0.3 Learning outcomes After completing this worksheet you should be able to: manipulate data from transformation practical work understand and calculate transformation efficiency convert between units. Introduction This task is aimed at giving practice with the maths skills of using appropriate units (MS 0.1) and using ratios, fractions and percentages (MS 0.3). You will have studied genetic engineering (Chapter 21 in your Student book) and will know that the introduction of modified plasmids into host bacteria is called transformation. Transformation efficiency is a measure of how effectively plasmid DNA is taken up by the host cells. The units are colony forming units (cfu) per g of plasmid DNA. A colony forming unit is a transformed bacterium. Since this will produce a visible colony when grown on an agar plate, the calculation depends on knowing the number of colonies that show evidence of transformation and the mass of DNA supplied to the bacteria at the start. A practical to demonstrate transformation efficiency is the transformation of bacteria using the green fluorescent protein gene. This task uses the efficiency of the transformation process as a context to practise the relevant maths skills. You will need to convert between units and apply dilution ratios. Worked example Question In an experiment: 1 25 g of DNA was re-suspended in 300 l of transformation solution l of this DNA suspension was transferred to a tube containing bacteria and a further 300 l of transformation solution. 3 Following a heat shock, 300 l of broth was added to the tube. This resource sheet may have been changed from the original 1

2 4 150 l of the resulting mixture was transferred to an agar plate containing antibiotic to kill any untransformed cells. 5 Following incubation, 78 transformed colonies were observed. a How much DNA was added to the bacteria in Step 2? b How much DNA was eventually spread on the plate in Step 4? c What is the transformation efficiency? Answer a First find the concentration of the DNA. 25 g of DNA was mixed with 300 l of transformation solution. So the DNA concentration was g l g l 1 Step 2 10 l of the 0.08 g l 1 DNA suspension was transferred. So the total transferred was g of DNA b First find the total volume in the transformation tube. 10 l were added to 300 l in Step 2 and a further 300 l were added in Step 3. So the volume in the transformation tube was l Step 2 Next work out the fraction of this volume that was transferred to the plate. 150 l was transferred out of a total volume of 610 l. So the fraction is (25% of the total volume). Step 3 Finally calculate the mass of DNA transferred. The 610 l contained 0.8 g of DNA. 25% of this was transferred to the plate. So the mass of DNA transferred is g. This resource sheet may have been changed from the original 2

3 c Transformation efficiency is found by dividing the total transformed colonies visible by the mass of DNA available. In this example there were 78 transformed colonies from 0.2 g of available DNA. Each colony corresponds with one transformed cell or colony forming unit (cfu). So the transformation efficiency is cfu g 1 DNA. Questions 1 Table 1 shows data from some transformation experiments. a Calculate the initial concentration of the rehydrated DNA for row a. b Calculate the mass of DNA transferred to the transformation tube in row b. c Calculate the mass of DNA transferred to the final plate in row c. (4 marks) d Calculate the transformation efficiencies for all four rows. (4 marks) This resource sheet may have been changed from the original 3

4 Mass of DNA at the start/ g used to rehydrate the DNA/ l of solution in the transformation tube/l of rehydrated DNA added to the transformation tube/l of broth added before plating/ l transferred to the agar plate/l Number of transformed colonies observed after incubation Example a b c d Table 1 2 In an experiment: ng of DNA was resuspended in 0.25 ml of transformation solution l of this DNA suspension was transferred to a tube containing bacteria and a further 250 l of transformation solution. 3 Following a heat shock, 0.25 ml of broth was added to the tube l of the resulting mixture was transferred to an agar plate containing antibiotic to kill any untransformed cells. 5 Following incubation, 81 transformed colonies were observed. a Calculate how much DNA was added to the bacteria in Step 2. Express your answer in ng. b Calculate how much DNA was eventually spread on the plate in Step 4. Express your answer in g. (3 marks) (3 marks) This resource sheet may have been changed from the original 4

5 c Calculate the transformation efficiency. Maths skills links to other areas Using small units and converting between them is a useful skill which may come up in questions relating to microscope image magnification calculations. Using ratios and fractions is also used in finding Rf values, making serial dilutions and in the Hardy Weinberg calculation. This resource sheet may have been changed from the original 5