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Biotechnology

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Title: Biotechnology


1
Biotechnology
13
2
Chapter 13 Biotechnology
  • Key Concepts
  • 13.1 Recombinant DNA Can Be Made in the
    Laboratory
  • 13.2 DNA Can Genetically Transform Cells and
    Organisms
  • 13.3 Genes and Gene Expression Can Be Manipulated
  • 13.4 Biotechnology Has Wide Applications

3
Chapter 13 Opening Question
  • How is biotechnology used to alleviate
    environmental problems?

4
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • It is possible to modify organisms with genes
    from other, distantly related organisms.
  • Recombinant DNA is a DNA molecule made in the
    laboratory that is derived from at least two
    genetic sources.

5
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • Three key tools
  • Restriction enzymes for cutting DNA into
    fragments
  • Gel electrophoresis for analysis and purification
    of DNA fragments
  • DNA ligase for joining DNA fragments together in
    new combinations

6
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • Restriction enzymes recognize a specific DNA
    sequence called a recognition sequence or
    restriction site.
  • 5'.GAATTC3'
  • 3'.CTTAAG5'
  • Each sequence forms a palindrome the opposite
    strands have the same sequence when read from the
    5' end.

7
Figure 13.1 Bacteria Fight Invading Viruses by
Making Restriction Enzymes
8
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • Some restriction enzymes cut DNA leaving a short
    sequence of single-stranded DNA at each end.
  • Staggered cuts result in overhangs, or sticky
    ends straight cuts result in blunt ends.
  • Sticky ends can bind complementary sequences on
    other DNA molecules.
  • Methylases add methyl groups to restriction sites
    and protect the bacterial cell from its own
    restriction enzymes.

9
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • Many restriction enzymes with unique recognition
    sequences have been purified.
  • In the lab they can be used to cut DNA samples
    from the same source.
  • A restriction digest combines different enzymes
    to cut DNA at specific places.
  • Gel electrophoresis analysis can create a map of
    the intact DNA molecule from the formed fragments.

10
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • DNA fragments cut by enzymes can be separated by
    gel electrophoresis.
  • A mixture of fragments is placed in a well in a
    semisolid gel, and an electric field is applied
    across the gel.
  • Negatively charged DNA fragments move towards the
    positive end.
  • Smaller fragments move faster than larger ones.

11
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • DNA fragments separate and give three types of
    information
  • The number of fragments
  • The sizes of the fragments
  • The relative abundance of the fragments,
    indicated by the intensity of the band

12
Figure 13.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 1)
13
Figure 13.2 Separating Fragments of DNA by Gel
Electrophoresis (Part 2)
14
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • After separation on a gel, a specific DNA
    sequence can be found with a single-stranded
    probe.
  • The gel region can be cut out and the DNA
    fragment removed.
  • The purified DNA can be analyzed by sequence or
    used to make recombinant DNA.

15
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • DNA ligase is an enzyme that catalyzes the
    joining of DNA fragments, such as Okazaki
    fragments during replication.
  • With restriction enzymes to cut fragments and DNA
    ligase to combine them, new recombinant DNA can
    be made.

16
Figure 13.3 Cutting, Splicing, and Joining DNA
17
Concept 13.1 Recombinant DNA Can Be Made in the
Laboratory
  • Recombinant DNA was shown to be a functional
    carrier of genetic information.
  • Sequences from two E.coli plasmids, each with
    different antibiotic resistance genes, were
    recombined.
  • The resulting plasmid, when inserted into new
    cells, gave resistance to both of the antibiotics.

18
Figure 13.4 Recombinant DNA (Part 1)
19
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Recombinant DNA technology can be used to clone
    (make identical copies) 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.

20
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Usually only a few cells exposed to recombinant
    DNA are actually transformed.
  • To determine which of the host cells are
    transgenic, the recombinant DNA includes
    selectable marker genes, such as genes that
    confer resistance to antibiotics.

21
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Most research has been done using model
    organisms
  • Bacteria, especially E. coli
  • Yeasts (Saccharomyces), commonly used as
    eukaryotic hosts
  • Plant cells, able to make stem cellsunspecialized
    , totipotent cells
  • Cultured animal cells, used for expression of
    human or animal geneswhole transgenic animals
    can be created

22
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Methods for inserting the recombinant DNA into a
    cell
  • Cells may be treated with chemicals to make
    plasma membranes more permeableDNA diffuses in.
  • Electroporationa short electric shock creates
    temporary pores in membranes, and DNA can enter.

23
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Viruses and bacteria can be altered to carry
    recombinant DNA into cells.
  • Transgenic animals can be produced by injecting
    recombinant DNA into the nuclei of fertilized
    eggs.
  • Gene guns can shoot the host cells with
    particles of DNA.

24
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • The new DNA must also replicate as the host cell
    divides.
  • DNA polymerase does not bind to just any
    sequence.
  • The new DNA must become part of a segment with an
    origin of replicationa replicon or replication
    unit.

25
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • New DNA can become part of a replicon in two
    ways
  • Inserted near an origin of replication in host
    chromosome
  • It can be part of a carrier sequence, or vector,
    that already has an origin of replication

26
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • 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 which
    can be selectable markers

27
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • 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. 13, p. 249
29
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • A plasmid from the soil bacterium Agrobacterium
    tumefaciens is used as a vector for plant cells.
  • A. tumefaciens contains a plasmid called Ti (for
    tumor-inducing).
  • The plasmid has a region called T DNA, which
    inserts copies of itself into chromosomes of
    infected plants.

30
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • 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.

31
In-Text Art, Ch. 13, p. 250
32
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Most eukaryotic genes are too large to be
    inserted into a plasmid.
  • Viruses can be used as vectorse.g.,
    bacteriophage. The genes that cause host cells to
    lyse can be cut out and replaced with other DNA.
  • Because viruses infect cells naturally they offer
    an advantage over plasmids.

33
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Usually only a small proportion of host cells
    take up the vector (1 cell in 10,000) 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
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • If a vector carrying genes for resistance to two
    different antibiotics is used, one antibiotic can
    select cells carrying the vector.
  • If the other antibiotic resistance gene is
    inactivated by the insertion of foreign DNA, then
    cells with the desired DNA can be identified by
    their sensitivity to that antibiotic.

35
Figure 13.5 Marking Recombinant DNA by
Inactivating a Gene
36
Concept 13.2 DNA Can Genetically Transform Cells
and Organisms
  • Selectable markers are a type of reporter genea
    gene whose expression is easily observed.
  • Green fluorescent protein, 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.

37
Figure 13.6 Green Fluorescent Protein as a
Reporter
38
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • DNA fragments used for cloning come from three
    sources
  • Gene libraries
  • Reverse transcription from mRNA
  • Products of PCR
  • Artificial synthesis or mutation of DNA

39
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • 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.
  • A vector is taken up by host cells which produce
    a colony of recombinant cells.

40
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • 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.
  • 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.

41
Figure 13.7 Constructing Libraries
42
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • DNA can be synthesized by PCR if appropriate
    primers are available.
  • The amplified DNA can then be inserted into
    plasmids to create recombinant DNA and cloned in
    host cells.
  • Artificial synthesis of DNA is now fully
    automated.

43
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • Synthetic oligonucleotides are used as primers in
    PCR reactions.
  • Primers can create new sequences to create
    mutations in a recombinant gene.
  • Longer synthetic sequences can be used to
    construct an artificial gene.

44
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • Synthetic DNA can be manipulated to create
    specific mutations in order to study the
    consequences of the mutation.
  • Mutagenesis techniques have revealed many
    cause-and-effect relationships (e.g., determining
    signal sequences).

45
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • A knockout experiment inactivates a gene so that
    it is not transcribed and translated into a
    functional protein.
  • In mice, homologous recombination targets a
    specific gene.
  • The normal allele of a gene is inserted into a
    plasmidrestriction enzymes are used to insert a
    reporter gene into the normal gene.
  • The extra DNA prevents functional mRNA from being
    made.

46
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • The recombinant plasmid is used to transfect
    mouse embryonic stem cells.
  • Stem cellsunspecialized cells that divide and
    differentiate into specialized cells
  • The original gene sequences line up with their
    homologous sequences on the mouse chromosome.

47
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • The transfected stem cell is then transplanted
    into an early mouse embryo.
  • The knockout technique has been important in
    determining gene functions and studying human
    genetic diseases.
  • Many diseases have a knockout mouse model.

48
Figure 13.8 Making a Knockout Mouse
49
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • Complementary RNA
  • Translation of mRNA can be blocked by
    complementary microRNAsantisense RNA.
  • Antisense RNA can be synthesized and added to
    cells to prevent translationthe effects of the
    missing protein can then be determined.

50
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • RNA interference (RNAi) is a rare natural
    mechanism that blocks translation.
  • RNAi occurs via the action of small interfering
    RNAs (siRNAs).
  • An sRNA is a short, double stranded RNA that is
    unwound to single strands by a protein complex,
    which also catalyzes the breakdown of the mRNA.
  • Small interfering RNA (siRNA) can be synthesized
    in the laboratory.

51
Figure 13.9 Using Antisense RNA and siRNA to
Block the Translation of mRNA
52
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • DNA microarray technology provides a large array
    of sequences for hybridization experiments.
  • A series of DNA sequences are attached to a glass
    slide in a precise order.
  • The slide has microscopic wells, each containing
    thousands of copies of sequences up to 20
    nucleotides long.

53
Concept 13.3 Genes and Gene Expression Can Be
Manipulated
  • DNA microarrays can be used to identify specific
    single nucleotide polymorphisms or other
    mutations.
  • Microarrays can be used to examine gene
    expression patterns in different tissues in
    different conditions.
  • Example Women with a propensity for breast
    cancer tumors to recur have a gene expression
    signature.

54
Figure 13.10 Using DNA Microarrays for Clinical
Decision-Making
55
Concept 13.4 Biotechnology Has Wide Applications
  • Almost any gene can be inserted into bacteria or
    yeasts and the resulting cells induced to make
    large quantities of a product.
  • Requires specialized expression vectors with
    extra sequences needed for the transgene to be
    expressed in the host cell.

56
Figure 13.11 A Transgenic Cell Can Produce Large
Amounts of the Transgenes Protein Product
57
Concept 13.4 Biotechnology Has Wide Applications
  • Expression vectors may also have
  • Inducible promoters that respond to a specific
    signal
  • Tissue-specific promoters, expressed only in
    certain tissues at certain times
  • Signal sequencese.g., a signal to secrete the
    product to the extracellular medium

58
Concept 13.4 Biotechnology Has Wide Applications
  • Many medically useful products are being made
    using biotechnology.
  • The two insulin polypeptides are synthesized
    separately along with the ß-galactosidase gene.
  • After synthesis the polypeptides are cleaved, and
    the two insulin peptides combined to make a
    functional human insulin molecule.

59
Figure 13.12 Human Insulin From Gene to Drug
(Part 1)
60
Figure 13.12 Human Insulin From Gene to Drug
(Part 2)
61
Concept 13.4 Biotechnology Has Wide Applications
  • Before giving it to humans, scientists had to be
    sure of its effectiveness
  • Same size as human insulin
  • Same amino acid sequence
  • Same shape
  • Binds to the insulin receptor on cells and
    stimulates glucose uptake

62
Concept 13.4 Biotechnology Has Wide Applications
  • Pharming Production of pharmaceuticals in farm
    animals or plants.
  • Example Transgenes are inserted next to the
    promoter for lactoglobulina protein in milk. The
    transgenic animal then produces large quantities
    of the protein in its milk.

63
Figure 13.13 Pharming
64
Concept 13.4 Biotechnology Has Wide Applications
  • Human growth hormone (for children suffering
    deficiencies) can now be produced by transgenic
    cows.
  • Only 15 such cows are needed to supply all the
    children in the world suffering from this type of
    dwarfism.

65
Concept 13.4 Biotechnology Has Wide Applications
  • Through cultivation and selective breeding,
    humans have been altering the traits of plants
    and animals for thousands of years.
  • Recombinant DNA technology has several
    advantages
  • Specific genes can be targeted
  • Any gene can be introduced into any other
    organism
  • New organisms can be generated quickly

66
Figure 13.14 Genetic Modification of Plants
versus Conventional Plant Breeding (Part 1)
67
Figure 13.14 Genetic Modification of Plants
versus Conventional Plant Breeding (Part 2)
68
Table 13.2 Potential Agricultural Applications
of Biotechnology
69
Concept 13.4 Biotechnology Has Wide Applications
  • Crop plants have been modified to produce their
    own insecticides
  • The bacterium Bacillus thuringiensis produces a
    protein that kills insect larvae
  • Dried preparations of B. thuringiensis are sold
    as a safe alternative to synthetic insecticides.
    The toxin is easily biodegradable.

70
Concept 13.4 Biotechnology Has Wide Applications
  • Genes for the toxin have been isolated, cloned,
    and modified, and inserted into plant cells using
    the Ti plasmid vector
  • Transgenic corn, cotton, soybeans, tomatoes, and
    other crops are being grown. Pesticide use is
    reduced.

71
Concept 13.4 Biotechnology Has Wide Applications
  • Crops with improved nutritional characteristics
  • Rice does not have ß-carotene, but does have a
    precursor molecule
  • Genes for enzymes that synthesize ß-carotene from
    the precursor are taken from daffodils and
    inserted into rice by the Ti plasmid

72
Concept 13.4 Biotechnology Has Wide Applications
  • The transgenic rice is yellow and can supply
    ß-carotene to improve the diets of many people
  • ß-carotene is converted to vitamin A in the body

73
Figure 13.15 Transgenic Rice Rich in ?-Carotene
74
Concept 13.4 Biotechnology Has Wide Applications
  • Recombinant DNA is also used to adapt a crop
    plant to an environment.
  • Example Plants that are salt-tolerant.
  • Genes from a protein that moves sodium ions into
    the central vacuole were isolated from
    Arabidopsis thaliana and inserted into tomato
    plants.

75
Figure 13.16 Salt-tolerant Tomato Plants (Part 1)
76
Figure 13.16 Salt-tolerant Tomato Plants (Part 2)
77
Concept 13.4 Biotechnology Has Wide Applications
  • Instead of manipulating the environment to suit
    the plant, biotechnology may allow us to adapt
    the plant to the environment.
  • Some of the negative effects of agriculture, such
    as water pollution, could be reduced.

78
Concept 13.4 Biotechnology Has Wide Applications
  • Concerns over biotechnology
  • Genetic manipulation is an unnatural interference
    in nature
  • Genetically altered foods are unsafe to eat
  • Genetically altered crop plants are dangerous to
    the environment

79
Concept 13.4 Biotechnology Has Wide Applications
  • Advocates of biotechnology point out that all
    crop plants have been manipulated by humans.
  • Advocates say that since only single genes for
    plant function are inserted into crop plants,
    they are still safe for human consumption.
  • Genes that affect human nutrition may raise more
    concerns.

80
Concept 13.4 Biotechnology Has Wide Applications
  • Concern over environmental effects centers on
    escape of transgenes into wild populations
  • For example, if the gene for herbicide resistance
    made its way into the weed plants
  • Beneficial insects can also be killed from eating
    plants with B. thuringiensis genes

81
Answer to Opening Question
  • Bioremediation is the use, by humans, of
    organisms to remove contaminants from the
    environment.
  • Composting and wastewater treatment use bacteria
    to break down large molecules, human wastes,
    paper, and household chemicals.
  • Recombinant DNA technology has transformed
    bacteria to help clean up oil spills.

82
Figure 13.17 The Spoils of War
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