Chapter 12 Study Guide PDF

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Chapter 12 Study Guide – “Biotechnology and Synthetic Biology” Learning Objectives Explain how to clone a gene of interest and how would you assay for the gene product? What gene would you choose to clone and why? Vocabulary Biotechnology Complementary DNA Gel Electrophoresis Genetically modified organism (GMO) Genetic engineering Green fluorescent protein (GFP) Hybridization Molecular cloning Nucleic acid probe Polymerase Chain Reaction (PCR) Polyvalent vaccine Recombinant DNA Reporter Gene Restriction enzyme Subunit vaccine Ti plasmid Transgenic organism Vector Vector vaccine Review Questions 1. How does PCR work and when would you use it? What is Taq? Where did it come from and why is that beneficial? a. PCR—polymerase chain reaction is DNA replication in vitro, multiplying segments of target DNA up to a billionfold during amplification (doubling) i. Thermocylcer—automated PCR machine ii. Requires DNA polymerase and artificial primers made of DNA iii. Amplifies stretches of a few kbp target from within a template 1. Denature template DNA by heating and add two DNA oligonucleotide primers in excess 2. DNA poly extends primers using template DNA 3. Heat to separate strands and cool 4. Repeated 20-30 times yielding 106 to 109 fold increase b. Taq is a thermostable DNA polymerase that is critical to PCR because it has resistance to high temperatures c. Applications i. Cloning or sequencing, phylogenetic studies, amplifying very small DNA quantities, medical diagnostics, forensic science

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ii. Reverse transcription PCR 1. Can make DNA from an mRNA template 2. Uses the enzyme reverse transcriptase to convert RNA into complementary DNA Why is PUC19 a great cloning vector? a. Molecular cloning—movement of a gene from original source to small and manipulatable genetic element (vector) i. Results in recombinant DNA ii. Gene can be manipulated iii. Cloned DNA replicated b. PUC19 i. Plasmid that is widely used ii. Restriction enzyme iii. Vector and foreign DNA cut with the enzyme iv. DNA ligase used v. Insertional inactivation of gene within lacZ distrupts gene and detecs cloned DNA vi. Transformants plated on media containing ampicillin and X-gal (turns blue) to detect Beta galactosidase activity vii. Cell containing vector and insert are white What is EcoRI? What is it used for? a. Restriction enzyme that produces sticky ends Explain how you could clone a gene for insulin production into E. coli. a. See last page What is a reporter gene? The product of which reporter gene yields a green color? a. Reporter genes encode protein easy to detect and assay i. May be used to report presence/absence of a genetic element in a vector ii. Can be fused to other genes or other gene promoters to study gene expression b. Green fluorescent protein (GFP) What is the advantage of using genetic engineering to make insulin? a. Clean reproduction b. Get what you want What is a transgenic plant? a. Transgenic—genetically engineered organism that contains a gene (transgene) from another organism b. Ti plasmid contains genes that mobilize DNA for transfer to plant; responsible for virulence i. T-DNA: plasmid segment transferred to plant; sequences at ends essential for transfer ii. Causes crown-gall disease (tumors on the roots) c. Bacteria transfers the plasmid that causes tumors What is rBST and how is it made? What is it used for? a. Recombinant bovine somatotropin—commonly used in dairy industry; stimulates milk production in cows b. Makes cows produce more milk c. The gene was transformed into an E. coli so that it produces rBST. Which is then injected into cows i. Has to be reverse transcriptase-ized before integrating

9. Explain why recombinant vaccines might be safer than some vaccines produced by tradition methods? a. Recombinant vaccines can: i. Modify a pathogen with genetic engineering to delete virulence factors and retain those that elicit immune response ii. Engineer genes from a pathogenic virus to genome of a harmless carrier virus iii. Polyvalent vaccine: single vaccine that immunizes against two different diseases 10. What are the important differences among a recombinant live attenuated vaccine, a vector vaccine and a subunit vaccine? a. Subunit vaccines contain only a specific protein or proteins from a pathogenic organism (ex. Coat protein of a virus) i. Popular bc large amounts of immunogenic proteins can be administered at high dosage with less risk than attenuated or killed vaccines b. Vector Vaccines induce immunity to a pathogen by way of a harmless carrier virus

Ex: Insulin Isolate the gene (whatever gene product you want, pick the gene for it) Pancreas cells—lyse them to extract the DNA Restriction enzymes cut up the DNA to get specific gene from DNA Blunt Ends (EcoRV—cuts in the same place on both strands so that the ends are the same) Sticky Ends (EcoRI—restriction enzyme isolated from E. coli; restriction site is where it cuts) Ideal because they have sticky ends (one piece of the DNA sticks off by itself with only one strand; can base pair with another single strand of DNA) DNA is in a test tube Gel Electrophoresis (separates DNA. Smaller pieces go farther, larger pieces don’t travel as far) Stain with Ethidium Bromide (mutagen that causes mutations) Put it on Trans-illuminator (UV light) Cut out bands (bands are the DNA) and put it in the tube PCR (polymerase chain reaction) in a thermocycler (amplification) (heat up so that DNA strands separate) Primers, ATCG, and Taq (DNA polymerase from a thermophile-- Thermas aquaticus) Taq copies the DNA 1. Denaturation by breaking H-bonds bw base pairs 2. Priming—incubated at low temp so that primers can attach 3. Extension—incubating at moderate temperature so that polymerase can rapidly replicate DNA Done for every band individually Puc19 (plasmid—dsDNA, circular, self-replicate) Has AMP (ampicillin resistance gene that enables you to grow it on ampicillin media) and LacZ Polylinker site—multiple places where multiple restriction enzymes will cut it Must cut EcoRI (used to cut) and plasmid (combine them with DNA ligase in a test tube) Sticky ends allow them to come together Want the Puc19 to pick up the insulin genes (we only need one)

Ligase seals them when they come together Transformation Experiment Competent E. coli (bc that is the bacteria we want to pick up the Puc19 plasmid with insulin gene) + plasmid Has to be made competent with cold calcium Blue-White Selection x-gal media with ampicillin (selects for gene that has the resistance) if it doesn’t grow then it doesn’t pick up the gene ? mutants blue—make Beta galactosidase white—mutants—make insulin recombinant insulin (bc made by bacteria using recombinant dna) Insulin replaced the lacZ portion of the gene...