Genetic Transformation

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Genetic Transformation
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Historical Perspective

• Frederick Griffith 1928 London
• First controlled demonstration of genetic
  transformation
• Griffith made the observation that nonpathogenic
  bacteria (Streptococcus pneumoniae) became
  pathogenic when mixed with a virulent strain of
  heat-killed S. pneumoniae (i.e. injected mixture
  killed mice)
• The mechanism of transforming
• nonpathogenic bacteria to deadly
• bacteria was not known
• In 1944 Oswald Avery demonstrated
• that DNA is responsible for conferring
• pathogenic properties

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What is Genetic Transformation?

• Genetically modification of a cell
• Involves uptake of foreign DNA
• Replication within organism
• Gene expression
• DNA RNA Protein
• Introduction of foreign DNA Terms to know
• By viruses Transduction
• Between bacteria Conjugation
• In mammalian cells Transfection

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Gene Cloning

• Amplification and isolation of a particular gene
  sequence
• Requires the generation of recombinant DNA (rDNA)
  
• i.e. combining DNA that does not naturally occur
• Insertion of the gene into a plasmid (circular
  DNA)
• Transformation of bacteria for replication
• Select for cells that have received the
  recombinant
• DNA
• Select individual colony for scale-up culture and
  replication of cloned DNA

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Cloning a Gene into a Plasmid
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Genetic Transformation into E.coli
Ampicillin resistance gene (Ampr) and target
gene on bacterial plasmid
Individual colony is selected and cultured to
amplify recombinant DNA
Plasmid enters some bacteria
Only E. coli containing plasmid survive on
Ampicillin plates
Cell division
Transformation mixture is plated on to agar plate
containing Ampicillin
Bacterial clones
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Key Steps for Transformation

• Bacterial cell suspension is placed in CaCl2
  solution
• Cells must be in log phase of growth.
• Cells are kept on ice until heat shock treatment
• Heat shock at 42 C for one minute
• Recover period in LB broth
• Cells are spread on appropriate selection plates

Protein of interest
Protein for antibiotic resistance
Plasmid DNA enters the bacterial cell and the
genes are expressed.
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Components of Gene Cloning

• Plasmid (to carry rDNA into cell)
• Enzymes
• Restriction enzymes for cutting vector and insert
• DNA ligase for joining DNA fragments
• Selection process

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Plasmids

• Small circular dsDNA separate from bacterial DNA
• Plasmids exist in bacteria, yeast, organelles
• Single or multiple plasmid copies per cell
• Easy to isolate and manipulate
• Used as vector for transforming bacteria with
  foreign DNA
• Foreign DNA is inserted after cutting with
  restriction enzymes
• Plasmids contain certain genes which offer a
  competitive advantage for bacteria (i.e.
  antibiotic resistance)
• Positive Selection confers growth advantage i.e.
  able to grow in presence of antibiotic
• Insert gene for expression (lt10kb insertion)

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Arabinose Operon

• Gene induction
• Arabinose operon
• Three structural genes araB, araA, and araD
  encode enzymes for arabinose metabolism
• Initiator region, araI contains both the operator
  and promoter
• The araC gene encodes an activator protein, AraC,
  which binds to initiator region

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Arabinose Operon Regulation

• Activation
• Arabinose binds the activator protein
• AraC/arabinose complex facilitates binding of RNA
  polymerase to the promoter which turns on the ara
  operon.
• Activation also depends on cyclic AMP
• Repression
• Without arabinose, AraC protein binds araI and
  araO regions forming a loop and preventing
  transcription of the ara operon
• Inducible promoter is used to control gene
  expression

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Competent Cells

• Competence is the ability of cells to take up
  exogenous DNA from the environment
• Two types of competence
• Natural competence Bacteria have cellular
  machinery to take up DNA from environment
• Artificial competence Cells are made competent
  in the laboratory allowing them to take up DNA

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Preparing Competent Bacteria

• Heat Shock
• Drives DNA into cells
• Hold cells on ice in presence of CaCl2 to promote
  permeability of cells to plasmid DNA
• Cells are heat shocked at 42 ºC for 50 60
  seconds to allow circular plasmid DNA to enter
  cells
• Electroporation
• Subject cells to electric shock to perforate
  membrane
• Plasmid DNA enters cells through temporary holes
• Efficient transformation of large plasmids

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Plant Transformation

• What is plant transformation
• Objective To transform the entire organism not
  individual cells
• Systemic infection of Arabidopsis
• by transformation of female gametes

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Genetic Engineering

• Involves
• Isolating genes
• Modifying genes for improved function
• Packaging gene for insertion into new organism
• Developing transgenes
• Development of organisms that express new traits
  not found in nature
• Extended shelf-life (produce)
• Herbicide resistance (Roundup Ready)
• Faster growth rate, larger
• Terms
• Transgene is a genetically engineered gene added
  to a species
• Transgenic refers to an organism containing an
  artificially introduced transgene (i.e. not
  through breeding)

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Agrobacterium tumefaciens

• Natural tool for plant transformation
• How it works tumor induction
• Transfer of DNA to plant

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Methods of Plant Transformation

• Agrobacterium
• Easiest and most simple
• Cut plant tissue in small pieces, soak in
  Agrobacterium suspension
• Some cells will be transformed by the bacterium
• Grow on selection medium (rooting or shooting)
• Some plants will not transform with the method
• Particle Bombardment
• DNA is coated onto gold or tungsten particles
• Particles are shot into young plant cells
• Low efficiency
• Most plants can be transformed
• Electroporation
• Electric shock induces transient holes in cell
  membranes
• DNA enters cells
• Viral transformation
• Use plant virus as vector to introduce DNA
• Not always integrated into plant genome

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Applications and Potential

• Genetically Modified Organisms
• Agriculture
• Health and Medicine
• Biotechnology
• Scientific Research
• Industry and Environment
• Gene therapy

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Genetically Modified Organisms (GMOs)

• GMOs
• Express traits not normally found in nature
• Result of introducing foreign DNA
• Highly controversial
• Safety concerns
• Environmental implications
• Can we blindly trust profit-driven industry?

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Agriculture

• Herbicide resistant crops
• Soybean, corn canola, lettuce, strawberry,
  potato, wheat
• Virus resistance
• Papaya resistance to papaya ringspot virus
• Golden rice
• Engineering rice to produce Vitamin A
• Edible vaccines in development
• Plant containing pathogen protein is ingested
• Body produces antibodies against protein
• Conferring resistance (ex diarrhea, hepatitis B,
  measles)
• Bananas, potato, tomato

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Health and Medicine

• Biotherapeutics
• Antibodies
• Hormone
• Enzymes
• Disease Indications
• Liver disease
• Genetic diseases
• Kidney disorders
• Digestive disorders
• Cancer
• Infectious disease

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Biotechnology

• Chymosin
• Genetically engineered enzyme
• Used for curdling milk productsin cheese
  production
• Revolutionized cheese production
• Previously rennin was isolated from newborn calf
  intestine (expensive, inhumane)
• Inexpensive, readily available
• Bovine somatotropin (bST)
• increased milk production in cows
• Other examples
• Insulin
• Interleukin
• Human growth hormone
• Interferon

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Scientific Research

• Protein production using genetic transformation
• Objectives
• Generate antibodies
• Assay development
• Structure determination
• Protein-protein interaction

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Industry and Environment

• Bioremediation Using bioengineered microbes to
  clean up pollution and contaminated sites
• Indicator bacteria Detecting pollution and
  contamination in the environment
• Waste management
• Sewage
• Petroleum products

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Gene Therapy Overview

• Viral vector is used to deliver genetic material
  to target cells (ex. liver, lung)
• The viral vector then injects the gene for a
  defective or missing protein
• The cell then produces the functional protein and
  restores the target cell to a normal state
• Viruses used for gene therapy
• Retroviruses
• Adenoviruses
• Adeno-associated viruses
• Herpes simplex viruses
• Gene therapy is experimental with poor success in
  clinical trials
• There are no FDA-approved gene therapy products
  on the market