Recombinant DNA and Biotechnology

1. Recombinant DNA and Biotechnology

2. 18 Recombinant DNA and Biotechnology • 18.1 What Is Recombinant DNA? • 18.2 How Are New Genes Inserted into Cells? • 18.3 What Sources of DNA Are Used in Cloning? • 18.4 What Other Tools Are Used to Study DNA Function? • 18.5 What Is Biotechnology? • 18.6 How Is Biotechnology Changing Medicine and Agriculture?

3. 18 Recombinant DNA and Biotechnology Bioremediation is the use of microorganisms to remove pollutants. Some microbes can digest some components of crude oil, but researchers are developing genetically modified organisms that can clean up oil more rapidly and effectively. Opening Question: Are there other uses for microorganisms in environmental cleanup?

4. 18.1 What Is Recombinant DNA? • Recombinant DNA is a DNA molecule made in the laboratory using at least two different sources of DNA. • Restriction enzymes and DNA ligase are used to cut DNA into fragments and then splice them together in new combinations.

5. 18.1 What Is Recombinant DNA? • The first recombinant DNA was made in 1973 using plasmids from E. coli. • This research was the start of recombinant DNA technology.

6. Figure 18.1 Recombinant DNA

7. 18.1 What Is Recombinant DNA? • Some restriction enzymes recognize palindromic DNA sequences: • 5′…….GAATTC……3′ • 3′…….CTTAAG……5′ • Some make straight cuts, others make staggered cuts, resulting in overhangs, or sticky ends.

8. 18.1 What Is Recombinant DNA? • Sticky ends can bind by base pairing to other sticky ends. • Fragments from different sources can be joined. • Then ligase catalyzes formation of covalent bonds between adjacent nucleotides at fragment ends, joining them to form a single, larger molecule.

9. Working with Data 18.1: Recombinant DNA • In 1973, the first recombinant plasmid was made using the restriction enzyme EcoRI and two plasmids with resistance to different antibiotics: • pSC101 had a gene for tetracycline resistance. • pSC102 had a gene for kanamycin resistance.

10. Working with Data 18.1: Recombinant DNA • Question 1: • In one experiment, some pSC101 was cut with EcoRI but not sealed with DNA ligase. • Cut or intact pSC101 were used to transform E. coli cells, which were grown on media containing tetracycline or kanamycin. • What can you conclude from this experiment?

11. Working with Data 18.1: Recombinant DNA

12. Working with Data 18.1: Recombinant DNA • Question 2: • In another experiment, pSC101 and pSC102 were mixed and treated in three ways:

13. Working with Data 18.1: Recombinant DNA • Did treatment with DNA ligase improve the efficiency of genetic transformation by the cut plasmids? • What is the quantitative evidence for your statement?

14. Working with Data 18.1: Recombinant DNA • Question 3: • How did the antibiotic-resistant bacteria arise in the “None” DNA treatment?

15. Working with Data 18.1: Recombinant DNA • Question 4: • Did the EcoRI + DNA ligase treatment result in an increase in doubly-resistant bacteria over controls? • What data provide evidence for your statement?

16. Working with Data 18.1: Recombinant DNA • Question 5: • For the EcoRI + DNA ligase treatment, compare the number of transformants that were resistant to either tetracycline or kanamycin alone to the number that were doubly resistant. • What accounts for the large difference?

17. Figure 18.2 Cutting, Splicing, and Joining DNA

18. 18.2 How Are New Genes Inserted into Cells? • Recombinant DNA technology can be used to clone, or make identical copies, of genes. • Transformation: recombinant DNA is cloned by inserting it into host cells (transfection if host cells are from an animal). • The altered host cell is called transgenic.

19. 18.2 How Are New Genes Inserted into Cells? • Usually only a few cells are transformed. • To determine which of the host cells contain the new sequence, the recombinant DNA includes selectable marker genes, such as genes that confer resistance to antibiotics.

20. 18.2 How Are New Genes Inserted into Cells? • The first host cells used were bacteria, especially E. coli. • Yeasts (Saccharomyces) are commonly used as eukaryotic hosts. • Plant cells are also used—they have the ability to make stem cells (unspecialized, totipotent cells).

21. 18.2 How Are New Genes Inserted into Cells? • Cultured animal cells can be used to study expression of human or animal genes. • Whole transgenic animals can also be created.

22. 18.2 How Are New Genes Inserted into Cells? • Inserting the recombinant DNA into a cell: • Cells may be treated with chemicals to make plasma membranes more permeable—DNA diffuses in. • Electroporation—a short electric shock creates temporary pores in membranes, and DNA can enter.

23. 18.2 How Are New Genes Inserted into Cells? • Viruses can be altered to carry recombinant DNA into cells. • Plants are often transformed using a bacterium that inserts DNA into plant cells. • Transgenic animals can be produced by injecting recombinant DNA into the nuclei of fertilized eggs.

24. 18.2 How Are New Genes Inserted into Cells? • The new DNA must also replicate as the host cell divides. • It must become part of a segment with an origin of replication—a replicon or replication unit.

25. 18.2 How Are New Genes Inserted into Cells? • New DNA can become part of a replicon in two ways: • Inserted near an origin of replication in host chromosome • Part of a carrier sequence, or vector,that already has an origin of replication

26. 18.2 How Are New Genes Inserted into Cells? • Plasmids make good vectors: • Small and easy to manipulate • Have one or more restriction enzyme recognition sequences that each occur only once • Many have genes for antibiotic resistance that can be used as selectable markers

27. 18.2 How Are New Genes Inserted into Cells? • Have a bacterial origin of replication (ori) and can replicate independently of the host chromosome • Bacterial cells can contain hundreds of copies of a recombinant plasmid. The power of bacterial transformation to amplify a gene is extraordinary.

28. In-Text Art, Ch. 18, p. 377 (1)

29. 18.2 How Are New Genes Inserted into Cells? • A plasmid from the soil bacterium Agrobacterium tumefaciens is used as a vector for plant cells. • Plasmid Ti (tumor inducing) causes crown gall. • The plasmid has a region called T DNA, which inserts copies of itself into chromosomes of infected plants.

30. In-Text Art, Ch. 18, p. 377 (2)

31. 18.2 How Are New Genes Inserted into Cells? • T DNA genes are removed and replaced with foreign DNA. • Altered Ti plasmids transform Agrobacterium cells, then the bacterium cells infect plant cells. • Whole plants can be regenerated from transgenic cells, or germ line cells can be infected.

32. 18.2 How Are New Genes Inserted into Cells? • Most eukaryotic genes are too large to be inserted into a plasmid. • Viruses can be used as vectors (e.g., bacteriophage). • Because viruses infect cells naturally, they offer a great advantage over plasmids.

33. 18.2 How Are New Genes Inserted into Cells? • Usually only a small proportion of host cells take up the vector, and they may not have the appropriate sequence. • Host cells with the desired sequence must be identifiable. • Selectable markers such as antibiotic resistance genes can be used.

34. 18.2 How Are New Genes Inserted into Cells? • Selectable markers or reporter genes: genes whose expression is easily observed. • There are several types: • Antibiotic resistance in a plasmid or other vector. A transformed host cell will grow on medium containing the antibiotic.

35. 18.2 How Are New Genes Inserted into Cells? • The lacZ gene codes for an enzyme that can convert the substrate X-Gal into a bright blue product. • If foreign DNA is inserted within the lacZ gene, and the plasmid transforms bacterial cells, they will not be able to convert X-Gal, and will produce white colonies. Untransformed cells produce blue colonies.

36. Figure 18.3 Selection for Recombinant DNA

37. 18.2 How Are New Genes Inserted into Cells? • Green fluorescent protein (GFP), which normally occurs in a jellyfish, emits visible light when exposed to UV light. • The gene for this protein has been isolated and incorporated into vectors as a reporter gene. • It has also been modified to produce other colors.

38. Figure 18.4 Green Fluorescent Protein as a Reporter

39. 18.3 What Sources of DNA Are Used in Cloning? • DNA fragments used for cloning come from four sources: • Gene libraries • Reverse transcription from mRNA • Products of PCR • Artificial synthesis or mutation of DNA

40. 18.3 What Sources of DNA Are Used in Cloning? • A genomic library is a collection of DNA fragments that comprise the genome of an organism. • The DNA is cut into fragments by restriction enzymes, and each fragment is inserted into a vector, which is used to produce a colony of recombinant cells.

41. Figure 18.5 Constructing Libraries

42. 18.3 What Sources of DNA Are Used in Cloning? • If bacteriophage λ is used as a vector, about 160,000 “volumes” are required to store the library. • One petri plate can hold thousands of phage colonies, or plaques. • DNA in the plaques is screened using specific probes.

43. 18.3 What Sources of DNA Are Used in Cloning? • Smaller DNA libraries can be made from complementary DNA (cDNA). • mRNA is extracted from cells, then cDNA is produced by complementary base pairing, catalyzed by reverse transcriptase.

44. 18.3 What Sources of DNA Are Used in Cloning? • mRNAs do not last long in the cytoplasm and are often present in small amounts, so a cDNA library is a “snapshot” of the transcription pattern of the cell. • cDNA libraries are used to compare gene expression in different tissues at different stages of development.

45. 18.3 What Sources of DNA Are Used in Cloning? • RT-PCR: reverse transcriptase and PCR are used to create and amplify a specific cDNA sequence. • This is used to study expression of particular genes in cells and organisms.

46. 18.3 What Sources of DNA Are Used in Cloning? • Artificial DNA with specific sequences can be synthesized by PCR. • The process is now fully automated and is used to create PCR primers and DNA with specific characteristics, such as restriction sites or specific mutations. • Fragments can be pieced together to form artificial genes.

47. 18.4 What Other Tools Are Used to Study DNA Function? • A way to study a gene and its protein: express it in cells that do not normally express the gene or in a different organism. • The gene must have a promoter and regulatory sequences for the host cell.

48. 18.4 What Other Tools Are Used to Study DNA Function? • Another way to study a gene: overexpress it so that more product is made. • A copy of the coding region is inserted downstream of a different, stronger promoter, and cells are transformed with the recombinant DNA.

49. 18.4 What Other Tools Are Used to Study DNA Function? • Mutations can be created in the laboratory in synthetic DNA. • Consequences of the mutation can be observed when the mutant DNA is expressed in host cells.

50. 18.4 What Other Tools Are Used to Study DNA Function? • Genes can also be studied by inactivating them (e.g., transposon mutagenesis) to define the minimal genome. • In animals, this is called a knockout experiment.