Prelim1 - BIOMI 404 Prelim 1 February 27, 2008 Question 1...

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BIOMI 404 Prelim 1 February 27, 2008 Question 1 1a) Recombinant-based in vivo expression technology is used to figure out which bacterial promoters are active during infection in vivo . A plasmid is made that carries pieces of bacterial genomic DNA fused to a reporter gene; in this article, the reporter gene is the resolvase gene ( tnrP ) that creates the sucrose-resistant selectable phenotype. The sucrose-resistant phenotype is created when resolvase excises a cassette flanked by res sites and containing neo-sacB. The resolvase gene is only transcribed, however, if a cross-over recombination event happens that puts tnrP within an active promoter or operon; the crossover happens by homologous recombination, and a second wild-type copy of the viral gene of interest exists so that a mutant phenotype is not created. Scientists can then isolate bacteria that were only sucrose resistant in vivo and sequence the viral DNA that was fused with the tnrP gene , making a transcriptional unit with tnrP . 1b) Signature-tagged mutagenesis works by taking a mutant pool of bacteria and introducing this pool to a mouse model. The bacterial mutant pool is created via transposon mutagenesis: a transposon with random oligomers (which serve as an individual tag) and with a selectable marker, such as antibiotic resistance, is introduced into the bacterial strain. The transposon inserts itself into the bacterial genome, making a specific mutant. Each colony made by this method represents a different mutation. Then, an animal is inoculated with a mixture of all the mutant, transposon-containing colonies. After infection, a specific organ is removed, and bacteria are recovered. Then, DNA is removed from colonies that grew, and PCR primers are used to amplify the tags for each transposon; the tags are also radiolabeled. The radiolabeled tags are then used to probe the original mutants. If a mutant does not hybridize with a tag, that means that the tag was not isolated, and that that specific mutant had a gene required for survival and infection of the host. The DNA around the transposon of the non-surviving mutant can than be analyzed to see which gene was interrupted. Advantages: Requires few animals, since many mutants can be studied within one animal. Disadvantages: Can only look at 96 mutants at a time (since the chips have 96 wells). Genes that are required for pathogenicity (but not survival) can be missed. Also, if a gene produces a toxin, this gene will also be missed. In vivo induced antigen technology (IVIAT) is used to figure out which host-produced antibodies are made to antigens produced in vivo only. This is done by first extracting serum from an infected patient, and also growing the bacterium in vitro . When you mix the patient serum and the in vitro -grown microorganism, patient antibodies will bind to bacterial antigens; any antibody that did not bind anything must bind antigen that is only produced in vivo . At the same time, an expression library must be made by cutting up the
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This note was uploaded on 05/11/2008 for the course BIO 4040 taught by Professor Debbie,d. during the Spring '08 term at Cornell University (Engineering School).

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Prelim1 - BIOMI 404 Prelim 1 February 27, 2008 Question 1...

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