Exam II 2009 - SECOND EXAM CHE 339/BIO 335/BME 339 FALL 2009 Tuesday YOUR NAME BID 1 The gene fraternity is thought to be responsible for a

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Unformatted text preview: SECOND EXAM CHE 339/BIO 335/BME 339 FALL 2009 Tuesday, November 10, 2009 YOUR NAME: BID: 1. The gene fraternity is thought to be responsible for a disorder characterized by complete lapse of mental function in young adult males, particularly following moderate alcohol consumption. [15 points total] Questions A, B, and C are independent of each other! A. The fraternity gene is expected to contain at least two introns. Describe how you would make a suitable library that will allow you to isolate the protein coding sequence of the gene. (5 points) B. You wish to clone the gene. Earlier studies at Texas A&M identified a small fragment of DNA that is known to be part of the FRATERNITY gene. Briefly describe (in three steps) how you would isolate the gene from the library you made in A. (5 points) C. After you clone the gene in bacteria you are asked to perform a Northern and a Southern blot in human cells. What are these and what will you learn from them? (5 points) 2. The gene for the mammalian pyruvate dehydrogenase enzyme has been cloned in E. coli. It was confirmed by hybridization experiments that the bacteria synthesize mRNA for the mammalian pyruvate dehydrogenase. However, no pyruvate dehydrogenase enzymatic activity was found in the recombinant cells. You know that the gene construct you are using has a Shine Dalgarno sequence. [10 points total] A. Suggest four possible explanations for this result. (6 points) B. For two of these possible explanations outline how you can go about fixing the problem. (4 points) 3. Describe (in one sentence each) four ways for introducing DNA into cells. [8 points] 4. Gene Synthesis [6 points]: A. These days it is very easy to make synthetic genes of up to a few thousand nucleotides in length. Describe briefly how this is done. (4 points) B. The process in A cannot be used to synthesize very long genes say 100,000 nt or longer. What process is used to make such long genes? (name- no need to describe) (2 points) 5. Bioseparations [15 points total]: You are assigned to purify an intracellular protein produced in Bacillus subtilis, a Gram-positive bacterium with a thick cell wall. Questions A-D are independent of each other! A. Describe two techniques you could use to break the cells without causing denaturation of the protein. (4 points) B. Next, you select to enr1cn the protein from the cell debris (DNA, cell wall fragments, etc.) by fractional ammonium sulfate precipitation. How does this work? (4 points) C. Afier B you have elected to use ion exchange chromatography and gel filtration or size exclusion chromatography. What does each of these steps accomplish and what order would you use them in? (4 points) D. How can you estimate the molecular weight ofthe protein? (3 points) 6. Anflnsen won me 1973 Chemistry Nobel prize for proposing that the correctly folded state of proteins is thermodynamically favored. This means that under physiological conditions the correctly folded state of a protein is the preferred conformation of the protein and this is in turn dictated sole by the amino acid sequence. However, it is also well known that under a variety of conditions proteins fail to fold properly, forming aggregates instead. Why does this happen? When protein aggregates form in bacteria what can be done to obtain the correctly folded (and firnctional) form ofthe protein? [8 points total] 7. Give three reasons why protein secretion from the cytoplasm is important for biotechnology. [6 points] 8. In one sentence for each explain the significance/utility of the following [2 points each; 14 points total] A. Alkaline Phosphatase B. Polyhydrin promoter C. Gene chip (DNA microarray) D. Dideoxynucleotide (think Sanger sequencing) E. Emulsion PCR F. Yeast Artificial Chromosome G. Signal Sequence 9. DNA sequencing [14 points] A. Describe three key applications of high throughput DNA sequencing (2 points each, 6 total) B. Describe briefly how DNA sequencing works (i.e. Sanger sequencing). (5 points) C. Finally describe the idea behind single molecule sequencing (the Helicos Biosciences technology) and the main advantage it offers. (3 points) Multiple choice questions from the Quizzes [1 point each] I. Sometimes proteins are separated by liquid-liquid extraction. This means that: A. B. C. D. First you add one organic solvent to separate the protein and then a second solvent. You mix the protein with water miscible organic solvents such as butanol. You generate an aqueous 2-phase solution by adding a polymer like PEG and a second component which could be either a salt or another polymer. You simply add starch to the protein solution and the starch separates from the proteins. II. The isoelectric point of a protein is: A. The potential that has to be applied during protein electrophoresis to keep the protein at a fixed position i.e. not to move into the gel. B. The value of the pH where all acidic aa are deprotonated i.e. in the C00- form. C. D. The potential that has to be applied for the protein to become oxidized. The value of the pH where the protein is the least soluble because it has no net charge. III. For recombinant protein production it is essential to use inducible promoters that can be turned on by the investigator because: A. B. C. Otherwise the cells would be producing protein continuously and would be growing more slowly. Recombinant protein synthesis is toxic to the cells and therefore it has to commence only when a certain level of cell mass has been reached in a culture. The synthesis of the recombinant protein shuts down the replication of the plasmid encoding the gene. All promoters are naturally inducible i.e. they require that transcription is turned on by some other molecule. It allows for better process control. IV. In 454 sequencing the incorporation of a base into a DNA that is being sequenced is detected by monitoring: A B. C. D. E. Fluorescence. Radioactivity due to the addition of a radio labeled nucleotide. Emission of a photon (light). By hybridization with probe DNA None of the above. ...
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This note was uploaded on 12/13/2011 for the course BME 339 taught by Professor Georgiou during the Spring '11 term at University of Texas at Austin.

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Exam II 2009 - SECOND EXAM CHE 339/BIO 335/BME 339 FALL 2009 Tuesday YOUR NAME BID 1 The gene fraternity is thought to be responsible for a

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