CHEM112B_Midterm (SP10)_Key

CHEM112B_Midterm (SP10)_Key -...

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chem-112b-key-midterm-S2010.docx 4/28/2010 Page 1 of 4 UCSD Chem 112B Midterm Answer Key, S2010 Instructors: T. Crowley & G. Ghosh Eight questions, 100 points. Show calculations where appropriate for chance at partial credit. 1) (8 pts) The genome of the E. coli DH5 α strain is a single, circular chromosome of ~4.7 megabase pairs. A plasmid construct is prepared with the coding sequence for a fluorescent protein (1 kb) in the multiple cloning sequence (MCS) of pUC18. An aliquot of E. coli DH5 α is mixed with the plasmid and the standard transformation procedure is performed. The bacteria are then plated on LB/ampicillin medium. Which of the following statements will be most accurate? Explain your reasoning. a) The bacterial chromosome will be replicated and the cells will proliferate regardless of whether they have the plasmid. b) Bacteria that have taken in the plasmid will proliferate and it is possible that each newly-arising cell will have multiple copies of the plasmid. c) Bacteria that have taken in the plasmid will proliferate, but each newly-arising cell will have only a single plasmid molecule because plasmid replication is coordinated with chromosomal replication. Statement b is most accurate. Plasmids have an origin of replication that allows their genome to be replicated independent of the host cell chromosome. Although the host cell DNA polymerase usually synthesizes the new DNA, copying of the plasmid is autonomous . Each E. coli cell has a single chromosome but it is possible that multiple copies of the plasmid may be present. 2) (12 pts) The enzyme DNase cleaves the ribose-phosphate backbone of double-stranded DNA. In the early days of recombinant DNA work, the nick translation technique was used to make radioactive copies of DNA templates in vitro . DNase was used at a relatively low concentration so that in most cases when the enzyme made contact, only one strand of the DNA was cleaved. A student prepares some plasmid DNA from a bacterial culture by the procedure you used in exercise 6. An aliquot is examined by electrophoresis immediately after the prep is completed and the remainder of the plasmid is stored at 4 0 C. One week later, a second aliquot is removed and another gel is run. Comparison of the gel images reveals a single band immediately after the prep was completed. After the one-week interval, a band is present at the same position as seen previously but there is also another band, migrating more slowly. What is the most likely explanation for these observations? Plasmid molecules are circular and in bacteria the circle is supercoiled. For a given number of kilobase pairs, the supercoiled configuration is much more compact than linear (generated by restriction digest at a single site) or relaxed (nicked) circle that lacks supercoiling. Therefore supercoil migrates ahead of the other two forms during electrophoresis. Supercoil is converted to relaxed circle by cleavage of the ribose- phospahte backbone on one strand. In the described scenario, the plasmid preparation probably
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This note was uploaded on 05/26/2010 for the course CHEM Chem 114C taught by Professor Hoffman during the Spring '07 term at UCSD.

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CHEM112B_Midterm (SP10)_Key -...

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