Bi1_2011_PS6

Bi1_2011_PS6 - Bi1 The Biology and Biophysics of Viruses...

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Bi1: The Biology and Biophysics of Viruses Spring 2011 Problem Set 6: Viruses, ELISA and Antibodies Due Wednesday, May 18 at 4:00 P.M. in the Bi 1 closet Name: ______________________________________________________________ Section # : ___________________________________________________________ Mail Code : ___________________________________________________________ TA Names : ___________________________________________________________ Date and Time turned in : _______________________________________________ Number of pages including this one : _____________________________________ AFTER YOU FINISH: How long did it take you to complete this problem set? ____________________________ Go to the Bi1 moodle site at http://courses.caltech.edu/ and take the homework survey There are 2 questions. The number of parts to each question is listed at the beginning of each; be sure to answer all the parts! Grade: Problem 1 __________ Problem 2 __________ TOTAL: _________ HOMEWORK INSTRUCTIONS 1) Turn in your homework stapled to this cover page. 2) Use separate sheets of paper for your answers. 3) Write or type your answers neatly. 4) Put your name on each page of your answers. 5) Box your answers, please, so that the grader can find them. Points may be deducted if you don’t follow these instructions!
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Page 2 of 10 Recommended reading for problem set: Chapter 49 (3/E and 2/E) with an emphasis on 49.2 (3/E and 2/E) Lectures 19 and 20 (Antibodies, Binding data, Flow cytometry) Problem 1: Gene delivery using a lentiviral vector and ELISA (60 points 13 parts) Please read ALL text and figure legends carefully. You are interested in studying mutants of 2G12, a broadly neutralizing anti-HIV IgG antibody that binds a carbohydrate epitope on the HIV envelope protein gp120. To facilitate your research, you would like to create a cell line that stably expresses and secretes wild-type 2G12 (i.e., unaltered 2G12) and cell lines that stably express and secrete the different 2G12 mutants you will construct. Here we will guide you through the steps a researcher might take to create a stable cell line expressing a protein of interest. We will describe the procedure for wild-type 2G12 the same procedure would be used to generate cell lines expressing each of the 2G12 mutants. Part I. A. (2 points) You have heard that some labs have used lentiviruses to deliver genes into cells. Give an example of a lentivirus and describe why lentiviruses might serve as useful vehicles for gene delivery. (≤ 3 sentences) B. (4 points) In order to make a lentivirus that includes a gene or genes of interest (called a recombinant lentivirus), you must first insert your gene(s) into a special plasmid vector, which we’ll call “Vector”. The plasmid contains a gene for a green fluorescent protein (GFP). After you’ve inserted the DNA that encodes 2G12 into Vector using the cloning methods you’ve learned about, the finished plasmid is call ed Vector-2G12. Below is a plasmid map. How many genes are required to encode an IgG antibody such as 2G12, and which part of the antibody do the gene(s) encode? Why is it useful for Vector-2G12 to include a GFP gene? (created with PlasMapper 2.0, Dong et al.)
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Page 3 of 10 C. Your next step is to introduce your plasmid into eukaryotic cells so that the 2G12 gene will be packaged into lentiviruses that are assembling in those cells. To accomplish this, you must introduce your plasmid together with a plasmid that encodes the gag , pol , and env genes, which are required for lentiviral assembly.
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