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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
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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
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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
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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
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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
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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
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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
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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
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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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