630 Microbiology for Teachers

Genetic Engineering and Biotechnology

Genetic Engineering uses Recombinant DNA Technology

• Deliberate modification of an organism's genetic information by directly changing its genome
• Accomplished by methods collectively known as recombinant DNA technology, the basic strategy of which is to move the gene of interest from one genome to another utilizing in vitro recombination of DNA molecules

Molecular Cloning

• Isolating and fragmenting DNA
  • DNA is isolated by extraction from organism of origin
  • DNA is fragmented by restriction enzymes (endonucleases)
    • restriction endonucleases are used to isolate and rearrange specific segments of DNA because they cleave DNA after recognizing specific sequences
    • they normally protect bacterial cells by destroying (restricting) phage or plasmid DNA
    • example - EcoRI cleaves both DNA strands between G and A in the sequence GAATTC, leaving complementary single-stranded "sticky ends" on each remaining double-helix
    • naming - the 3 letters at the beginning indicate the name of the bacterium from which the enzyme was isolated, the next letter indicates the strain, and a roman numeral is added to distinguish multiple enzymes with the same origin and indicates their order of discovery (in this case (Escherichia coli, strain R, isolate I)
  • DNA fragments are separated by agarose electophoresis
• Introducing DNA fragments into a cloning vector
  • Vectors are autonomously replicating DNA units into which DNA fragments are inserted for cloning (increasing in number) and for transfer of genes (contained in fragments of DNA) from one cell to another
    • plasmids - independently replicating circular pieces of DNA; useful for working with small (up to 5,000 base pairs = k kbp) pieces of DNA
    • bacteriophages - pieces of DNA (up to 15 kbp) are incorporated into l-phage DNA, packaged in phage capsids and introduced into the host bacterial cell by transduction
    • cosmids - plasmids made up of the two "ends" of lambda-phage (useful for pieces of DNA up to 50 kbp)
  • Methodology
    • cut vector DNA with same restriction enzyme DNA fragment was cut with
    • anneal DNA fragment and vector via their "sticky ends"
    • use ligase to join them with covalent bonds (phosphodiester bonds)
• Transferring the recombinant DNA molecule into a host organism so it can be replicated
  • transformation is the transfer of naked DNA into cells that are rendered competent by treatment with calcium ions
  • electroporation uses electric charge to help permeate the host so that the DNA is taken up
• Detecting and purifying the clone
  • Protein encoded by a gene that is expressed in the host organism can be detected by its activity (if it is an enzyme) or by antibodies specific for it
  • DNA can be detected using nucleic acid probes (necessary if the protein is not expressed or the gene product is unknown)
• Expanding the clone by producing large numbers of cells or bacteriophage containing it

Other Methods or Tools Used in Genetic Engineering

• Southern blotting - technique for identification of DNA fragments (genes, etc.) based on their sequence
  • DNA fragments are separated by agarose electophoresis
  • Fragments are then transferred (blotted) onto a sheet of nitrocellulose
  • Fragments are identified by hybridization (due nucleotide complementarity) with radioactive probe
• Sequencing - determining the sequence of nucleotides in a fragment allows
  • verification of the identity of a DNA fragment
  • determination of the "meaning" of the information encoded in it
• PCR - polymerase chain reaction
  • amplifies specific gene (DNA) sequences
    • DNA is heated to separate the strands, then excess primers (one for each end of the sequence to be amplified) are added and annealed to the "target" DNA
    • primer extension gives a copy of the target DNA
    • cycles of heating, primer annealing and primer extension yield up to a billion copies of the target DNA molecule
  • yields large amounts of genes for cloning, sequencing or mutgenesis purposes
• Site-directed mutagenesis
  • mutation of a specific DNA base pair in a gene
  • one approach is as follows:
    • clone gene into a single-stranded vector
    • add synthetic oligonucleotide with one base mismatch
    • extend single strand with DNA polymerase
    • transform product into host
    • clone and select mutant
  • PCR can also be used for site-directed mutagenesis

Practical Applications

• Agricultural - transgenic animals and plants
  • animals - increasing growth rates via transfer of genes from fast-growing, but less popular food animals into slow-growing, popular food animals (e.g., carp genes into trout or salmon)
  • plants - insertion of genes for resistance to pesticides, for nitrogen-fixation, for amino acid synthesis and utilization
• Industrial - production of proteins (enzymes, etc.) used in manufacturing processes
• Environmental biotechnology - generating microbes for remediation (oil-eaters, etc.)
• Medical - production of antibiotics, insulin, growth hormone, interferon, clotting factor VIII, vaccines, probes for infectious and genetic disease diagnosis, gene therapy, genetics and regulation research

Social impact

• Safety
  • concern has been expressed (and rightly so) about release and uncontrolled replication of genetically-engineered microbes into the environment
  • therefore, great care is taken to "disable" these microbes by engineering into them metabolic defects that can only be overcome (enable growth) by providing growth factors
• Ethical considerations - these techniques have great potential for harm to the environment, to people, etc. if they are purposefully used as "killers" (e.g., for biological warfare)