02 January 2020

1. 02 January 2020 Today’s Title: CW: Genetic engineering case studies – 1 human insulin Learning Question: how can insulin be manufactured?

2. Aims from specification (n) outline the process involved in the genetic engineering of bacteria to produce human insulin;

3. Key words • Genetic engineering • Restriction enzymes • Bacterial plasmids • Antibiotic resistance • Transformation • Altered plasmids • Transgenic • Bacteriophage • Replica plating • Reverse transcriptase • cDNA

4. Reverse transcriptase • Enzyme that retroviruses carry to convert their RNA into DNA – the reverse of transcription • Scientists have used this to build copies of human genes from isolated mRNA • Single-stranded copy of DNA is formed from mRNA

5. Genetically engineering insulin • Stages to isolate the gene • Remove mRNA from β-cells in islets of langerhans • Incubate mRNA with reverse transcriptase • Produces complementary single stranded DNA • This is converted to double stranded DNA – insulin gene • DNA polymerase and supply of free nucleotides added to single stranded DNA to produce a copy of the original DNA called cDNA • Unpaired nucleotides are added to each end of the cDNA to create sticky ends complementary to cut plasmid

6. Genetically engineering insulin • Plasmids cut open with restriction enzymes are mixed with cDNA genes • DNA ligase seals plasmid with new gene – now a recombinant • Plasmids mixed with bacteria, some of which take up new plasmids, but not all • Bacteria grown on agar plates to create colonies of clones

8. Not all bacteria take up plasmid  • Some bacteria do not take up plasmid • Some bacteria take up plasmid that has not sealed with new cDNA and resealed with out it • Some will take up recombinant plasmid – these are transformed bacteria  • Can’t tell the different just by looking at them!

9. Identification of transformed bacteria by replica plating • Plasmids are chosen that carry genes that make them resistant to 2 different antibiotics (ampicillin and tetracycline) • These resistant genes are known as genetic markers • The bacteria E.Coli are susceptible to both these antibiotic chemicals • Plasmids are cut by restriction enzymes that has its target site (where it cuts) in the middle of tetracycline resistance gene • This means that if the required gene is taken up, the gene for tetracycline resistance is broken up and fails to work, but the gene for ampicillin resistance does still work

11. How replica plating is used • Bacteria are grown on nutrient agar so all bacteria grow, forming colonies • Some cells are transferred onto agar that contains ampicillin, so only those that have taken up the plasmid with the gene to kill the bacteria will grow • Some cells from these colonies are transferred onto agar that has been made with tetracycline, so only those that have ten up a plasmid that does not have insulin will grow (the gene has not been cut)

12. How replica plating is used • By keeping track of which colonies are which, it can be deduced where the insulin gene is. • Bacteria growing on ampicillin agar, but not on tetracycline agar must contain the plasmid for the insulin gene • Colonies can be identified and grown on a larger scale • Bacteria producing insulin are harvested for later use

14. Questions A scientist has genetically engineered some bacterial cells to contain a desired gene and a gene that gives resistance to penicillin. The cells were grown on agar place and then transferred to a plate containing penicillin. • Explain why the scientist thinks colony A contains transformed bacterial cells, but colony B does not. (2) • Explain how the scientist might have inserted the desired gene into the plasmid (3) • Explain why being able to take up plasmids is useful to bacteria (2)

15. Answers • Colony A has grown on the agar plate containing penicillin (1 mark) so it contains the penicillin-resistance marker gene, which means it contains the transformed cells (1 mark) • The plasmid vector DNA would have been cut open with the same restriction endonuclease that was used to isolate the DNA fragment containing the desired gene (1 mark). The plasmid DNA and the gene (DNA fragment) would have been mixed together with DNA ligase (1mark) DNA ligase joins the sugar-phosphate backbone of the two bits of DNA (1 mark) • It is useful for bacteria to take up plasmids because the plasmids may contain useful genes (1 mark) that increase their chance of survival (1 mark)

16. One product manufactured using microorganisms is insulin. The process involves genetically engineering bacteria to synthesise human insulin. • (i) Describe how the isolated human insulin gene is inserted into a bacteria plasmid. • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • [4] • (ii) Suggest two ways in which the bacteria which take up the modified plasmids can be identified. • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • ......................................................................................................................... • [2] • [Total 6 marks]

17. plasmid cut by restriction enzyme; at specific sequence; same enzyme as used to cut (insulin) gene; sticky ends / described; ref. complementary sticky ends; ligase seals (sugar-phosphate) backbone / AW; max 4 • (ii) credit any two from the following: • 1 antibiotic resistance (gene) introduced and survivors have plasmid; • 2 fluorescent marker (gene) introduced and glowing bacteria have plasmid; • 3 identify bacteria producing insulin using antibodies; 2 • [6]