Chapter review…

1. Chapter review… Gene technology Ethics DNA recombinant technology Gene library Application Genetic engineering Gene cloning Genomic library cDNA library Transgenic organism DNA finger printing tools Genetically modified organism Target gene Cloning vector Restriction enzyme Modifying enzyme Host cell Polymerase chain reaction methods

2. Tools… DNA (gene) source DNA cloning vector DNA ligase Restriction enzyme Host cell

3. At the end of the lesson, you should be able to : • Describe the steps in gene cloning by using plasmid as the vector.

4. Concepts of molecular cloning The process of inserting a piece ofDNA molecule of interestinto aDNA carrier (vector) in order to make multiple copies of the DNA of interest in a host cell such asbacteria.

5. Concepts of molecular cloning • Purposes of molecular cloning : • Separate a gene from the other genes • Amplification of modified forms of genetic materials • Manipulation of a piece of DNA for further experiments

6. Concepts of molecular cloning • Methods in genetic engineering may be divided into three groups : • Plasmid method • Vector method • Biolistic method • Plasmid method is the most familiar and frequently used techniques in cloning process.

7. Steps in gene cloning Step 1 Isolation of gene Step 2 Cleave/cut Step 3 Insertion Step 4 Transformation and amplification Step 5 Screening

8. Overall procedures

9. Step 1Isolation of gene

10. Isolation of gene • Two different sources are selected : a. Gene of interest. • ( eg : gene coding for human insulin, etc) b. Plasmid (as vector)

11. Isolation of gene

12. Isolation of gene • The source of plasmids typically E. coli. • This plasmid carries two useful genes : • Ampr- conferring resistance to the antibiotic ampicillin • LacZ - encoding the enzyme beta-galactosidasewhich catalyzes the hydrolysis of sugar.

13. Isolation of gene • The plasmid has a single recognition sequence, within the lacZ gene, for the restriction enzyme used.

14. Step 2Cleave

15. Cleave Preparing plasmid vector • Treat and splice DNA donor (human) and plasmid DNA bacterial using the same restriction enzyme (eg : Bam HI in this case). • Bam HI cuts the same site on both molecules (palindromic) 5’- GGATCC - 3’ • The ends of the cut have an over hanging piece of single-stranded DNA that called ‘sticky ends’.

16. Cleave Preparing plasmid vector 5’ G A A T T C 3’ C T T A A G one DNA fragment another DNA fragment G A A T T C 3’ 5’ 5’ C T T A A G 3’

17. Step 3Insertion of rDNA

18. Insertion of rDNA Insertion of rDNA • In this case, both DNA prepared have complimentary sticky ends and thus can pair with each other when mixed. • This process called ‘annealing’ and use DNA ligase (sealing enzyme) to bring these things together.

19. Insertion of rDNA • Finally, this step formed a molecule of recombinant DNA/ recombinant plasmid (rDNA) / R-plasmid

20. Insertion of rDNA DNA fragments + enzymes recombinant plasmids host cells containing recombinant plasmids

21. Step 4Transformation & Amplification

22. Transformation & Amplification • Genetically engineered plasmids are re-introduced into the bacterium. • E. coli bacteria are made to take up these plasmid, but only 1% are found to contain rDNA. • So, rDNA must be located.

23. Transformation & Amplification Recombinant plasmid Calcium chloride (cold) + + Host cell To make the bacterial cell wall permeable to plasmid heat Treat the host cell with heat and presence of CaCl2 make the bacterial cell wall permeable to plasmid

24. Transformation & Amplification • Some of the recombinant plasmids enter the cytoplasm of the bacteria, this process formed genetically engineered bacteria. • Bacterial cells take up the recombinant plasmids by transformation.

25. Step 5 Screening

26. Screening • Genes are ‘expressed’ when their base sequence is being transcribed into mRNA for protein synthesis. • Manygenes do so only when activated(‘switch on’).

27. Screening • This creates a diverse pool of bacteria : • some bacteria that have taken up the desired r-plasmid DNA • other bacteria that have taken up other DNA, both recombinant and non-recombinant.

28. Screening • The vector used is a plasmid that carries two genes for antibiotic resistance. • Fortunately, plasmids often carry genetic information that makes bacterium resistant to certain antibiotics.

29. Screening • So, we can grow or culture the bacterial population in the presence of that antibiotic. • Only bacteria that have taken up genetically altered plasmids will resist the antibiotic and survive. • The bacteria that have not taken up plasmid, will die (because they are not resistant to the antibiotic)

30. Screening • In this case, plasmid that is used for this process is r-plasmid. • This r-plasmid has a gene for ampicillin resistance and a gene for tetracycline resistance. • Bam HI is used to cut in half the tetracycline resistant gene whilst the ampicillin resistant gene is uncut. • So, resistance to tetracycline antibiotic is lost because it is inactivated.

31. Screening

32. Screening • Bacteria, E.coli (host cell) is cultured on nutrient agar. • Only bacteria containing recombinant plasmid/ rDNA can grow in a plated nutrient agar with antibiotic ampicillin. • This is because the ampicillin resistant gene in the bacteria is still intact (uncut) & cause the bacteria to grow in medium containing ampicillin.

33. Screening • We can plate out the transformed bacteria on solid nutrient medium containing ampicillin and a sugar called X-gal. • Only bacteria that have the ampicillin-resistance plasmid will grow. • The X-gal in the medium is used to identify plasmids that carry foreign DNA.

34. Screening • Bacteria with plasmids lacking foreign DNA stainblue when beta-galactosidase hydrolyzes X-gal. • Bacteria with plasmids containing foreign DNA are white because they lack beta-galactosidase. • These bacteria are LacZ - unable to hydrolyze lactose.

35. Screening • In the final step, we will sort through the thousands of bacterial colonies with foreign DNA to find those containing our gene of interest.

36. Screening Carry gene of interest Carry other foreign gene