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Q1:BIOL1181 2250 Assignment 1: Applied Theory (WEEKS 1-3) ● Please read the instructions that have been posted on CANVAS before completing this assessment. Please use either Calibri (Font Size 11) or Arial Font (Size 10) to type your answers. Adhere to the space provided for each question. Question 1. You have recently joined a biotech company that specialises in the cloning and expression of human hormones for therapeutic use [Total = 12 marks]. Your first task it to express the Human Growth Hormone, whose structure is depicted below: Genomic DNA 5 5' size (bp) cDNA GH protein BIOL1181 Assignment 1: Applied Theory (WEEKS 1-3) Total Marks = 35 nukleotides 5175 5234 I exons Answer: ▼ IVS 1 Answer: 10 261 5'NT 5495 | ✓ 161 5655 5865 5984 6077 | ✓ ✓ IVS 2 ✓ IVS 3 209 120 93 31 40 55 IV 6241 165 • 65 IVS 4 253 6495 1 ▼ -3-23 1-31 32-71 72-126 127-191 (aa) V 195 6689 (21) You need to provide your supervisor with a strategy to clone this gene in an expression vector for expression in a bacterial cell system. You need to: 3'NT Poly A a. Explain to your supervisor which starting material you will use (genomic DNA? mRNA?) to prepare your insert, and why? [2 marks]. b. List the elements that must be present in this vector for the successful expression of the bacterial system, and why these elements are required? [4 marks]. BIOL1181 2250 Assignment 1: Applied Theory (WEEKS 1-3) c. List the mains experimental steps required for cloning your insert of interest into the vector, starting from extraction of the relevant nucleic acid from cells (as you have defined under Question 1a) and finishing with a pure population of bacteria that has acquired the plasmid. Indicate which enzyme(s) is(are) are required at each step [6 marks]? You can use a schematic. Answer:See Answer
Q2:Question 2. You have managed to express your his-tagged protein --Congratulations! [Total = 11 marks]. a. Which chromatography technique will you use to purify this His-tagged protein? [1 mark] Answer: b. Please explain to your supervisor how you will do it? (what are the steps in this technique?) [3 marks]. Answer: c. Unfortunately, when you run your protein on a gel, you see that a set of very large proteins co-purified with your His-tagged HGH (see picture below). You want to get rid of this very large protein. Which chromatography technique do you suggest to your supervisor can be used to solve this problem [1 mark]? Answer: 250 100 10 Contaminant proteins (>250 kDa) His-tagged HGH (22kDa) d. Explain the principle of the technique to your supervisor [3 marks]. Answer: e. You have now a purified protein - Well done! You assess its specific activity using the table below (testing the activity of your protein on a mammalian cell proliferation assay). Step Volume (ml) Crude bacterial extracts After your first step (Question 4a) After you second step (Question 4b) 500 1 1 Total protein (mg) Total HGH activity (units) 20,000 10 5 1,000 900 800 Specific activity (units/mg of protein) 0.05 i) What is the specific activity of your purified protein (bottom right cell in the Table)? [1 mark]. Answer: 90 ii) Using the numbers in this table, by which factor have you purified the specific activity of your protein between the start (crude extract) and the final step? [1 mark]. Answer: iii) How much (in mg) of the large, contaminating protein in the figure above did you remove by doing your second step? [1 mark]. Answer:See Answer

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