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Unformatted text preview: SECOND EXAM CHE 339/BIO 335/BME 339 FALL 2010 Thursday, November 11, 2010 YOUR NAME _________________________________ 1. Cloning and Expression Vectors (20 points total) A. What kind of vector would you use for the following purposes ( 1 points each ): (i) Expression of a gene encoded by a 20,000 bp fragment in E.coli: lambda phage (ii) Expression of a 4,000 bp fragment in yeast: plasmids (iii) Cloning of a human gene cluster totaling 150,000 bp into yeast: YAC B : Define in one sentence each, the following: ( 2 points each ) Ligase Enzyme used to ligate two dna fragments Klenow fragment of DNA polymerase Fragment of DNA polymerase I; allow for making blunt end fragments Electroporation: Use of high voltage to transport DNA into cells cDNA cloning: Cloning of mRNA b cDNA for recombinant protein expression β-galactosidase α− fragment complementation: Portion of LacZ gene for selection or protein stabilization plasmid: circular vector with ori, selectable markers, and multiple cloning sites C . What is a Bacmid? How are Bacmids used for protein expression and why? ( 5 points ) - Shuttling vector - Propagate in E. coli - Expression in Insect cells - Polyhydrin promoter for highly efficient expression - Close to mammalian post-translational modifications 2. What is the T7 promoter system and why is it used? (5 points)- Two gene system where: Inducible promoter + T7 RNA polymerase T7 promoter + GOI - Highly efficient at expression 3. Expression (20 points) NOTE: Questions are INDEPENDENT FROM EACH OTHER IF YOU CANNOT ANSWER ONE PART YOU MAY STILL BE ABLE TO ANSWER OTHER PARTS You wish to clone and express a human protease in bacteria. An antibody is available for the human protease....
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- Spring '11