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Name ____________________________________ Due Monday April 2,2012 No late assignments excepted!!! AP BIOLOGY • BIOTECHNOLOGY EXAM You are a molecular biologist working at a biotech company. Your CEO has identified an untapped niche market — Canadian bachelor farmers — and thinks the company can make millions developing new crops to sell to them. Your assignment is to develop, for the pickle industry, frostresistant cucumbers that glow fluorescent green so they are easy to find in the dark (short days in northern Canada, you know). You have identified the Northern cod as a fish species that produces the appropriate anti-freeze protein (AFP). You are also able to buy a plasmid from BioRad Laboratories containing the gene from a jellyfish species that produces a glowing fluorescent protein (GFP). 1. You obtain Arctic cod and raise them in an aquarium in the lab. Your first step is to make a genomic DNA library from the cod. Describe this process. Be specific! 2. Now you want to find the AFP gene in this cod genomic DNA library. Although the genome of the Arctic cod has not been sequenced, from careful study of GenBank, the online database of  DNA sequences, you are able to create a probe for the AFP gene from other arctic fish species. Explain what a probe is, and describe how you will use this to isolate the AFP gene from the DNA library. Be specific! 3. You have now isolated the bacterial colony which contains the plasmid with a DNA fragment that includes the cod AFP gene. As a research control, you need to double check the size of that cod DNA fragment. Describe a procedure for cutting out the cod DNA fragment from the plasmid and measuring its size. Cut out DNA fragment using restriction enzymes. 4. Once you isolate the bacterial colony containing the plasmid with the cod AFP gene, you need to combine this gene with the jellyfish GFP gene on a single plasmid. As is standard practice in the biotech industry, the GFP plasmid has a multiple cloning site and a selectable marker. Describe this cloning process. Be specific! 5. In order to patent your frost-resistant, fluorescent cucumbers, you need to submit a complete sequence of this recombinant plasmid (with the AFP and GFP genes) to the U.S. Patent Office. Describe the sequencing procedure you will follow. Be specific! 6. Research is going well! You are now working on overcoming the obstacle of getting your recombinant plasmid into the pickling cucumbers and getting high expression of both AFP and GFP. Unfortunately, one night someone breaks into your lab and steals your most promising prototype cucumber seedlings. Your company’s security force calls in the CSI New York team and they find a single hair follicle at the crime scene. How will they use this to find the thief? Be specific! 7. The theft has slowed research progress, but your CEO has come up with another great business idea! Now that you have isolated the AFP gene, you can produce the protein in bulk to be used as a cosmetic ingredient in cold weather moisturizing creams. Once again, your company will make millions selling the creams to Canadian bachelor farmers who spend so much time in the drying winter winds picking your glowing cucumbers in the snow. Rather than grinding up tons of cod fish and extracting AFP, you want to produce AFP economically in // E. coli  // . As a knowledgeable molecular biologist (you even got a 5 on your AP   Bio exam!), you understand that the bacteria will not be able to produce a functional anti-freeze protein from the original codfish genomic DNA clone. Explain why this is true. Also explain how to isolate a sequence from the fish that will allow bacteria to produce functional anti-freeze protein. Be specific! EXTRA CREDIT 1. After your company releases the frost-resistant, green-glowing cucumbers to the market, some customers complain that this new variety of pickles makes them burp too much. You are concerned this is cutting into market share. You wonder whether the interaction of the two new genes (AFP and GFP) is causing other proteins to be expressed in the cucumbers that induce burping. What technique would you use to investigate changes in gene expression between normal and AFP/GFP cucumbers? Do you also have to take into account the cold temperatures under which this new variety is grown? _____________________________________________________________________________ _____________________________________________________________________________  _____________________________________________________________________________  _____________________________________________________________________________  _____________________________________________________________________________  _____________________________________________________________________________