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HW4_2011(3) - BME 339 CHE 339/BIO 335 Fall 2011 Homework#4...

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Unformatted text preview: BME 339/ CHE 339/BIO 335 Fall 2011 Homework #4 Assigned: September 22, 2011 Due: Tuesday, October 18, 2011 1. From Shuler and Kargi, Bioprocess Engineering. 2. The enzyme NcoI recognizes the sequence 5' ‐C^C A T G G‐3' 3' ‐G G T A C^C‐5' It is used to cut the genome of a bacterium whose genome consists of 65% G or C and 35% A or T. The genome of the bacterium is 2,800,000 bp. How many fragments would you estimate to be generated by digestion of the genomic DNA with NcoI? 3. In the following paragraphs, numbers in bold type and in parentheses refer to questions given at the bottom. You may want to refer to the map of pBR322 that is attached. Professor Ivan Idea has decided to clone the gene elixir. He partially digests chromosomal DNA with the enzyme BamH1 (reaction does not go to completion, and therefore not all DNA sites that are recognized by the enzyme are digested) (1), isolates those DNA fragments that are approximately 10 kb; He cuts the plasmid vector pBR322 with BamH1 and inactivates the enzyme (2), and ligates the plasmid DNA to his chromosomal DNA fragments. He transforms the resulting mixture of plasmids into E. coli selecting for cells to become ampicillin‐resistant (AmpR), and screens them for being tetracycline‐sensitive (TetS) (3). He makes a replica plate on a nitrocellulose filter, denatures the DNA, fixes it to the filter and probes with radioactive mRNA for elixir (4). One positive clone was found that, following restriction mapping, was 15 kb (pBR322 is 4.5 kb). One of the enzymes used in mapping was BamH1 ‐ it produced 5 fragments of 5.5, 4.5, 3.0, 1.5, and 0.5 kb. Professor Ivan Idea now wanted to subclone the gene. He completely digested the plasmid with BamH1, inactivated the enzyme, and then performed a ligase reaction and a transformation, selecting, as before, for AmpR and TetS (5). After transferring to a filter and probing as before, analysis of positive clones showed that most plasmids were either 5.0 or 7.5 kb (6). Explain the rationale for (1), (2), (3), (4), and (5), and the result of (6) MAP OF pBR322 ...
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