706_S2006_PS1 - 7.06 Problem Set One, 2006 1. (Note: This...

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1 7.06 Problem Set One, 2006 1. (Note: This problem is based on the background knowledge in molecular biology, genetics, and biochemistry that you are presumed to have as a student coming into 7.06.) A recent study done in Schizosaccharomyces pombe (fission yeast) showed that the CHZ1 gene is involved in metabolizing the tasty bright orange cheese-flavored powder on Cheetos as an alternative carbon source. A chz1 knockout in S. pombe is inviable on medium in which Cheeto powder is the only carbon source. Intrigued by this novel metabolic pathway, you perform a BLAST search and find a CHZ1 homolog in your organism of choice, Saccharomyces cerevisiae (budding yeast). Your first goal is to knock out CHZ1 in budding yeast and examine the phenotype. a) Describe how you would make a knockout of CHZ1 in S. cerevisiae. b) You plate your chz1 mutant strain of Saccharomyces cerevisiae on medium in which Cheeto powder is the only carbon source, but the strain manages to grow and there is no obvious phenotype. Propose possible explanations for your finding. c) You decide to search for other genes involved in Cheeto powder metabolism. Luckily, because you work in S. cerevisiae , you have a very important tool available to you – the S. cerevisiae deletion collection. This is a collection of thousands of yeast strains, each of which lacks a single non-essential gene from the genome. Using the S. cerevisiae deletion collection as a starting point, describe how you would perform the genetic screen of interest to you. d) Next, you want to study the CHZ1 gene product in a more biochemical fashion. In order to purify lots of Chz1p protein, you create a DNA construct that will express a His-tagged version of Chz1p protein in bacteria. What is “His-tagged Chz1p” and how and why is making a construct that expresses this protein in bacteria useful to you? You express a His-tagged version of Chz1p in bacteria and purify the protein on an affinity column that has affinity for histidine. You elute the column with increasing amounts of imidazole (see the structure of imidazole below). e) Why does it make sense to elute the affinity column in this way?
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2 You run samples from all the fractions that eluted from your affinity column on an SDS-PAGE gel and find that all your Chz1p protein eluted quite nicely in fraction #15. However now your Chz1p protein is in a solution of fairly concentrated imidazole, and you want to remove it from that solution. You decide to purify your Chz1p away from the imidazole using a gel filtration column. f) Why and how would gel filtration work nicely to separate Chz1p away from the imidazole? You take fraction #15 from your affinity column, and run it over a gel filtration column. You collect the fractions from the gel filtration column, and analyze them in two ways. First, you follow the presence of protein in each of the fractions that elute off of your gel filtration column by its UV absorption spectrum, and second, you run each fraction on an SDS-PAGE gel and
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706_S2006_PS1 - 7.06 Problem Set One, 2006 1. (Note: This...

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