Exam II 2011 - CHE 339/BME 339/BIO 335 Exam II Thursday...

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CHE 339/BME 339/BIO 335 Exam II Thursday, November 10, 2011 Your name and uteid ________________________________ ANSWERS IN PENCIL CANNOT BE SUBMITTED FOR REGRADE Part A: Recombinant DNA (25 points) You are trying to find a gene that you named mfg1 ( m y f avorite g ene 1, just to be clear). This gene has lipase activity, it degrades triglycerides. You expect the gene to have introns. 1. Describe how you would make a suitable library from cell extract that would allow you to isolate the protein coding for this gene (5 pts). 2. You are now preparing to isolate the gene. Previous research has identified a small DNA fragment that is contained by the mfg1 gene. Briefly describe how you would isolate the gene from the library (5 pts).
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3. Your gene isolation is successful and you find a single gene sequence that you believe is mfg1 based on your analysis. You want to clone into a high expression plasmid (pBR322). You choose to use BamHI and SalI restriction enzymes. Outline the steps you will take using traditional cloning methods to clone in pBR322 (15 pts).
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Part B: DNA Analysis and sequencing (16 pts) 4. Which of the following methods can be used to determine the amount of a specific mRNA (e.g. mfg1 ) in cells (2 pts): a) Northern blotting. b) Southern blotting c) High throughput sequencing of cDNA d) All of the above (A, B and C). e) A and C 5. Describe four key applications of high throughput DNA sequencing (4 pts). 6. Describe the workflow of Roche 454 sequencing (10 pts).
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Part C: Protein engineering: expression, activity, and scale up (25 pts) You have successfully isolated the mfg1 gene that codes the lipase enzyme. This specific MFG lipase is only efficient at high pH and high temperature. 7. You desire an active MFG lipase enzyme that can then be added to laundry detergents to remove stains at low temperatures to improve energy efficiency. Lipase activity can be screened in media (solid or liquid) containing triglycerides and rhodamine b.
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Exam II 2011 - CHE 339/BME 339/BIO 335 Exam II Thursday...

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