Biotechnology

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 ß-galactosidase
• Treat plasmid with restriction enzyme
• Restriction enzyme makes cut in middle of
  ß-galactosidase gene

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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 ß-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 ß-galactosidase gene turn blue
• Colonies with gene inserted in middle of
  ß-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
  plants 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
  ß-carotene
• ß-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.