Exploring Biotechnology: Plasmids, Recombinant DNA, and Cloning in Genetic Engineering

1. Biotechnology Chapter 17

2. Biotechnology • Generally implies the genetic manipulation of organisms to give them new capabilities or improved characteristics • “bio” – life • “technology” – application of science to creation of products for human use, processes, and services

3. Plasmids • Discovered in 1960s • Small pieces of DNA • Separate from main bacterial chromosome • Generally not required for survival of bacterial cell • May carry genes that help cell survive in unusual environments • May carry information about antibiotic resistance

4. Plasmids • Can be replicated in cell just like main chromosome • Useful because easy to purify and work with • Have fewer genes than main chromosome • More stable in test tube • Easier to analyze • Bacterial cells can be induced to take up plasmids from surrounding solution • Process called transformation

5. Recombinant DNA • Microbiologists discovered in 1960s that bacteria contain enzymes capable of cutting DNA at specific base sequences • Restriction endonucleases or restriction enzymes • Function to protect cell by restricting invasion of cell by foreign DNA • Different restriction enzymes recognize different sequences of bases in DNA

6. Recombinant DNA • Restriction enzymes • Allow scientists to cut purified plasmid DNA in specific, reproducible places • Cuts can be reversed • Many make cuts with sticky ends • Overlapping regions of complementary DNA strands • At lower temperatures, ends stick together, and DNA can be covalently connected (ligated) using DNA ligase

7. Recombinant DNA • Can combine DNA pieces from different sources because sticky ends formed by particular restriction enzyme all have same base sequence • Forms recombinant DNA molecule • If process inserts new gene and DNA molecule becomes circular, new gene can be taken up with plasmid by receptive bacterium

8. Recombinant DNA • Key to genetic engineering is selecting desired combination of ligated pieces of DNA through procedure known as cloning

9. Cloning • Clone • Colony or group of cells or organisms • All members of group have same genes • Cloning • Replication of cells in the colony • Simple method of separating and eventually characterizing individual molecules of DNA • Individual molecule inserted into single bacterial cell can be replicated many times as cell divides • Cells in colony makes hundreds of thousands of copies of the same molecule

10. Cloning • Cloning example • Recombinant DNA molecules formed from plasmid and specific gene • Plasmid (pUC19) has two genes • Gene for resistance to ampicillin • Gene for making enzyme β-galactosidase • Treat plasmid with restriction enzyme • Restriction enzyme makes cut in middle of β-galactosidase gene

11. Cloning • Add new gene cut with same enzyme and ligate • Combine mixture of DNA molecules with suspension of bacterial cells in way so that each cell takes up only one DNA molecule • Spread bacteria on Petri dish containing nutrient agar, ampicillin, and chemical that turns blue in presence of β-galactosidase • Bacteria without plasmid will not grow on medium • Ampicillin kills cells

12. Cloning • Bacteria with plasmids (ampicillin resistance) survive and grow into colonies • Colonies with β-galactosidase gene turn blue • Colonies with gene inserted in middle of β-galactosidase gene remain white • Check white colonies to verify that they contain desired gene

13. Reverse Transcriptase and cDNA • Reverse transcriptase • Enzyme that can produce DNA using RNA template • Extract mRNAs and reproduce base sequences in DNA molecules • Starting with • extracted mRNA • a “primer” (small piece of DNA complementary in base sequence to mRNAs) • substrates (nucleoside triphosphates)

14. Reverse Transcriptase and cDNA • Reverse transcriptase adds nucleotides to primer to form • Single strands of DNA with base sequences complementary to mRNA templates • Result is mixture of “complementary” or “copy” DNAs • Abbreviated cDNAs

15. Polymerase Chain Reaction • PCR • Method to produce multiple copies of desired gene • Reaction combines • cDNAs with oligonucleotides (serve as “primers”) • Nucleoside triphosphates • DNA polymerase • Enzyme that synthesizes DNA

16. Polymerase Chain Reaction • Flexible technique • Can be used to • Detect traces of animal or plant genes in criminal investigations • Synthesize a gene with added restriction sites at ends • Useful for transforming plants • Allows gene to be inserted into plasmid and cloned in bacteria

17. Polymerase Chain Reaction • Steps in reaction cycle • Heat reaction solution almost to boiling • Separates complementary strands of DNA • Each strand is potential template • Cool reaction solution • Allows primers to bind to ends of any DNA with complimentary base sequences

18. Polymerase Chain Reaction • Heat reaction solution to optimum temperature for DNA polymerase • Allows synthesis of new DNA by addition of nucleotides to primers

19. Genomics • Genome • Genetic material in a cell • Genomics • Study of genome structure, function and evolution • Provides information useful in identifying genes • Genes with similar functions have similar base sequences

20. Genomics • Information obtained also teaches how networks of genes are regulated

21. Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • Scientists focused on condition called crown gall disease • Caused by Agrobacterium tumefaciens • Bacteria attach to plant cell walls and cause cells to begin dividing • Plant cells continue to divide even after bacteria have been killed with antibiotics

22. Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • Shows bacteria transform plant cells • Turns off normal mechanism for limiting cell division • Result much like an animal cancer • Mechanism involved • Infectious strains of A. tumefaciens have large plasmid, Ti (tumor-inducing) plasmid

23. Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • Bacterium injects part of plasmid into plant cells • Region injected (T-DNA) contains three genes that cause cells to divide and grow • Two genes code for enzymes that make auxin • One gene codes for a cytokinin (isopentenyl adenine) • Another gene is for enzyme that synthesizes amino acid called an opine • Opines out leak into intercellular spaces • Bacteria growing in intercellular spaces of tumor make enzyme allowing them to take up and metabolize opines

24. Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • In order to use Ti plasmid to carry genes into plant cells • Begin with T-DNA that has lost genes for auxin and cytokinin synthesis • Will not cause tumors in plant • Insert gene of interest • Controlled by promoter that regulates when and in what tissues it is turned on, and “reporter” gene that allows selection for cells that incorporate T-DNA • Recombinant T-DNA, usually in form of miniplasmid, transferred to A. tumefaciens cell with Ti plasmid lacking its own T-DNA

25. Insertion of Genes Into Plant Cells Using Agrobacterium tumefaciens • Spread on cut surface of piece of leaf • Bacteria transfer recombinant T-DNA to plant cells • Transfer leaf to medium containing antibiotics to kill bacterial cells • Engineers then select for plant cells that have incorporated reporter gene in T-DNA • Regenerate new plants using tissue culture techniques • Plants with new genetic information  transgenic plants

26. Biolistics • Method for adding new genetic material to plant cells • Uses gene gun • DNA containing gene is absorbed onto surface of small particles (subcellular-sized) of gold or tungsten • Particles pressed onto front of bullet • Loaded into gun • Fired at plant tissue

27. Biolistics • Metal plate with hole smaller than bullet stops bullet • Particles penetrate cells • Absorbed DNA dissolves into cell cytoplasm • Used as template for RNA synthesis • Genetic information expressed

28. Electroportation • Another method for getting DNA into plant cell • Based on discovery that short, high-voltage charge of electricity can produce temporary holes in plasma membrane without permanently harming cell

29. Electroportation • Make protoplasts by removing cell walls from recipient plant cells • Place protoplasts between two electrodes in ice-cold solution that contains the DNA • A few pulses of electricity produce membrane holes • Some DNA enters cells

30. Electroportation • Culture protoplasts under proper conditions • Protoplasts regenerate cell walls • Start dividing • Regenerate whole plants that express genes of DNA that entered protoplasts

31. Use of Viruses to Inject Genes Into Plants • Method does not produce permanently transformed plant • Viral and introduced genes not incorporated into plant’s nuclear DNA • Genes are not passed to seed formed by infected plant • Proteins made by infected plant in response to introduced genes • Often very useful

32. Applications of Biotechnology • Examples of proteins produced through genetic engineering • Insulin • Somatotropin • Erythropoietin • Clotting factors • Interferon

33. Applications of Biotechnology • Enzymes produced from genetically engineered bacteria (or yeasts) • Laundry detergent additives • Restriction enzymes • DNA polymerases

34. Applications of Biotechnology • Plants are being genetically engineered to produce vaccines • Designing and testing food plants that contain genes for proteins from pathogens • Banana (Musa sapientum) • Makes protein from hepatitis B vaccine • Alfalfa (Medicago saliva) sprout • Contains part of the cholera toxin

35. Development of New Plant Varieties • Produced plants with additional enzymes in anthocyanin pathway • Results are flowers with unusual colors or patterns • Hope to produce blue rose

36. Pest Resistance • Classical genetic techniques • Inefficient • Require many cycles of back crossing and selection • Modern molecular techniques • Use of Bacillus thuringiensis to control pests • Bacterium B. thuringiensis produces protein toxin that kills insects • Gene for toxin inserted into important crop plants • Potato, tomato, corn, cotton • Plants synthesize toxins and kill insects that graze on them

37. Pest Resistance • Insertion of gene for viral coat protein of tobacco mosaic virus TMV infects plants such as tomato, potato, eggplant, green pepper • Insertion of gene into these plants makes plant resistant to infection by virus • Development of crops resistant to herbicides • Resistant crop allows farmer to use herbicides to kill weeds in middle of field of crop plants • Allows more discriminating use of safer herbicides

38. Improved Quality of Fruit After Harvest • Large portion of harvested crops never reach consumers due to spoilage • First bioengineered food approved in United States • “FlavrSavr “ tomato • Contains gene that blocks synthesis of polygalacturonase (needed to soften tomato as it rots) • Lack of enzyme delays senescence (aging)

39. Improved Quality of Fruit After Harvest • Genes inserted into cantaloupes reduce synthesis of ethylene (ripening hormone)

40. Improved Nutrition • Some dietary staples are not most nutritious • Example: corn low in essential amino acids lysine and tryptophan • High lysine varieties of corn have been developed • Varieties of rice developed • One type produces seed with endosperm rich in β-carotene • β-carotene precursor for vitamin A • Help prevent blindness due to this deficiency

41. Improved Nutrition • Another type of rice rich in ferritin • Help prevent iron deficiency which results in anemia • Modification of canola (Brassica napus) • Given gene for fungal enzyme phytase • Enzyme phytase improves nutrition when included in feed for pigs and chickens • Releases phosphate from phytic acid • Helps animals grow faster and stronger

42. Improved Tolerance to Environmental Stress • Resistance to some stresses thought to depend on several genes • Research directed toward identifying genes that differ between stress-tolerant and stress-sensitive varieties

43. Is Biotechnology Safe? • Scientific issues to be evaluated in the approval of a genetically engineered food • Does the product contain any new allergenic material that might affect especially sensitive groups? • Are new toxic compounds introduced into the food supply, or are existing toxins increased to unacceptable levels?

44. Is Biotechnology Safe? • Are nutrient levels adversely affected? • Will the use of genes for antibiotic resistance (used to indicate when a plant has been stably transformed) compromise the use of important therapeutic drugs?

45. Is Biotechnology Safe? • Environmental effects • Impact of new plants on wildlife • Possibility that new genes from desired recipient species could be transferred to a related wild, weedy species • Concern when new gene confers protection against natural pests or chemical herbicides

46. Is Biotechnology Safe? • Field of biotechnology is growing • Research is key • The more we understand about plant and animal physiology and ecology, the more safely and effectively we can use biotechnology to improve our lives.