1. List and describe these tools of biotechnology: selection, mutation, restriction enzymes, agarose gel electrophoresis, cloning vectors, PCR
    • Biotechnology: use of microorganisms to make a product
    • Selection: Culture naturally occurring microbes that produce a desired product
    • Mutation: use mutagens to cause mutations that might result in a microbe with a desirable trait
    • Restriction enzymes: cut DNA at specific points, crucial for recombinant DNA.  Highly specific.
    • Agarose gel electrophoresis: Used as DNA fingerprinting
    • Cloning vectors: Self-replicating DNA used to carry the desired gene into a new cell
    • PCR: makes multiple copies of a piece of DNA
  2. Explain the significance of these to genetic engineering: vectors and restriction enzymes
    • Restriction enzymes recognize and cut DNA at a highly specified nucleotide sequence.  Two fragments of DNA from difference sources can be spliced together as long as they are cut by the same restriction enzyme.
    • Vectors carry the desired gene into the new cell, typically in plasmid form.  Restriction enzymes allow it to insert itsself
  3. Explain stepwise how bacterial recombinants are created and detected
    • Foreign DNA inserted into plasmid
    • Recombinant pasmid inserted into bacterium
    • Recombinant plasmid and bacterial chromosome both cut with same restriction enzyme
  4. List and explain the two sources of genes for genetic engineering
    • Gene libraries: contain either natural copies of genes or cDNA copies of genes made from mRNA (eukaryotes)
    • Synthetic DNA: Made by a DNA synthesis machine
  5. List and describe the different methods of introducing recombinant DNA into cells (including the Ti plasmid)
    • Transformation: treat cell at low temperature, increase temperature to "shock" DNA into cell
    • Electroporation: Create pores in cell, DNA enters through pores
    • Protoplast fusion: bacterial cell walls digested and protoplasts fused, cell wall formed by recombined cell
    • Microinjection: glass needle injecting DNA into cell
    • Gene gun: DNA-containing bullet shot into plant cell
    • Ti plasmid: Agrobacterium inserts the Ti plasmid into the plant cell
  6. List and discuss the importance of each component of a typical plasmid cloning vector
    • Origin of replication: so plasmid and reproduce in new cell
    • Selectable markers: to be able to distinguish which bacteria have your plasmid
    • Multiple cloning sites: where you insert the piece of DNA
  7. Describe how blue-white selection works in detail
    • Bacteria spread on NA plate containing ampicillin and x-gal. 
    • Only bacteria that picked up the plasmid will grow in the presence of ampicllin. 
    • All cells that grow (blue or white) have received plasmid
    • Cells that grow white ALSO have foreign DNA (what we are after)
    • Cells that grow white still have the ability to hydrolize x-gal and produce an indigo compound
  8. List and describe the different types of organisms used to make recombinant gene products
    • E. coli: easily grown, genome is known, cells lysed to get product (may contain endotoxin)
    • Bacillis subtilis: preferred in industry, secretes products, no endotoxins
    • Saccharomyces cerevisiae: genome is known, products secreted
    • Mammalian/human cells: harder to grow, make products for medical use (hormones, cytokins, interferons)
    • Plant cells: easily grown, produce plant alkaloids, isoprenoids
  9. Describe examples of the different applications of genetic engineering: terapeutic, scientific, agricultural
    • Therapeutic: insulin, subunit vaccines, DNA vaccines, gene therapy, gene silencing (RNAi)
    • Scientific: understanding of DNA, genome sequencing, DNA fingerprinting (track infectious diseases, forensic science, paternity test)
    • Agricultural: Bt toxin (degrades pests, but no effect on plants/vertebrates), herbicide resistant plants, supression of genes, animal growth hormones
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