1. Gene technology - what is it? - what is it used for? - how does it work?

2. How would you treat a diabetes sufferer? Where does the insulin come from?

3. Overview Genetic engineering involves the transfer of the DNA of one organism into the DNA of another. Several stages involved: –Isolating required gene e.g. insulin gene –Inserting the gene in a ‘vector’. –Transformation – the gene is delivered into the required cell for protein growth –Identification of host cells that have taken up the gene –Grow cells with new gene on a large scale.

4. Isolation of gene Method 1 Enzyme used: Reverse transcriptase. - RNA is taken from a cell that produces the required protein -The enzyme reverse transcriptase is found in retroviruses like HIV. It catalyses a reaction in which complementary DNA (cDNA) is made from mRNA + DNA nucleotides. The result is a single strand of cDNA. -DNA polymerase and free nucleotides are used to produce a double strand of cDNA. Stage One:

5. Isolation of gene Method 2 Enzyme used: Restriction endonuclease Gene can be removed from the chromosome using restriction enzymes (restriction endonucleases). Different restriction enzymes cut the DNA at a different base sequence. This is called a recognition sequence. Restriction enzymes are made by bacteria. They are used to destroy the DNA of bacteriophages (viruses that infect bacteria).

6. Sticky ends Vs Blunt ends Some endonucleases cut across the double strand of DNA in a straight line. This produces ‘blunt ends’:

7. Sticky ends Vs Blunt ends Some endonucleases cut across the double strand of DNA in a zig zag line. This produces ‘sticky ends’: What do you notice about the sequence?

8. Splicing a gene into a vector (in vivo) Insertion of the required gene into the cell that will make the protein (e.g. a bacterial cell). A vector is a ‘gene carrier’. Plasmids from bacteria are often used. Plasmids are short circular DNA strands. Stage Two:

9. Splicing a gene into a vector Plasmid must first be cut using the same restriction enzyme used to remove the required gene. Why? To produce complementary sticky ends to the required gene.

10. Cut genes & cut plasmids are mixed together. Under the right conditions, the sticky ends of the gene join with the sticky ends of the plasmid. A ligase enzyme catalyses this process (called ligation) Plasmid containing a human gene is called recombinant DNA (rDNA).

11. Review 1.Which enzymes can be used to isolate gene fragments? (2 marks) 2.Briefly explain how both of them isolate the fragments (6 marks) 3.What is commonly used as a ‘gene carrier’? (1 mark) 4.Explain the significance of ‘sticky ends’ in gene fragments (2 marks) 5.Name the enzyme that helps to join the gene fragment with the gene carrier (1 mark)

12. Transformation: Transferring rDNA to host cell Host bacterial cells containing rDNA are called transgenic organisms. Many methods have been tried to introduce rDNA into the host bacterial cell. Successful method: 1. Soak bacteria in ice-cold calcium chloride solution containing recombinant plasmids 2. Incubate for 2 mins at 42˚C 3. Bacterial cells take up recombinant plasmids Stage Three: Difficult process – uptake of plasmid by bacterial cells can be as low as 0.0025% Some plasmids may have also closed up before they took the gene fragment in.

13. Identification : Finding the GM bacteria with the plasmids + new gene Some bacteria will have taken up plasmids that DO NOT contain the desired gene – why? These need to be identified & destroyed so only the bacteria with the desired gene are cultured and grown. Several options using other useful genes on the plasmids (gene markers): –Antibiotic resistance genes –Genes that make particular enzymes –Genes that produce fluorescent proteins. Stage Four:

14. Gene markers – fluorescent proteins Genes that code for fluorescent proteins (e.g. green fluorescent protein – GFP -from jellyfish) can also be spliced into a plasmid. The desired gene is placed in the plasmid in the middle of the GFP gene. What will the effect of this be on the GFP? How can this be used to identify the plasmids & bacteria that contain the desired gene?

15. Gene markers – enzymes Like before, a gene that produces an enzyme can be introduced into the plasmid e.g. a gene that produces lactase. Lactase will turn its colourless substrate blue. How could is be used to identify which bacteria have successfully taken up the plasmid + desired gene?

16. Gene markers – antibiotic resistance Technique called replica plating. This time a gene for antibiotic resistance is cut in order to make space for the desired gene (e.g. tetracycline resistance) The bacteria are grown on agar plates containing tetracycline. What will happen to these bacteria? How is this problem overcome?

17. Replica plating Cells that contain the plasmids are grown on agar plates. Each cell will grow into a separate colony. A small sample from each colony is taken and grown on another (replica) plate. All colonies are carefully placed in the same spot as the original plate. Replica plate contains tetracycline – what happens to the new colonies that contain the desired gene? Colonies that did NOT grow on the replica plate must therefore contain the desired gene. The colonies from the original plate can now be used to grow the bacteria on a large scale.

18. Identification : Finding the GM bacteria with the plasmids.Produce a story board/flow diagram to explain and summarise how antibiotic resistance genes can be used to identify host cells containing the desired plasmids. Stage Four: