Name:_KEY_________________________ 1 2006 7.012 Problem Set 4 KEY Due before 5 PM on FRIDAY, October 27, 2006. Turn answers in to the box outside of 68-120. PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT. 1. You are studying a specific gene in yeast, and you want to express that yeast gene in E. coli. Your task is to design a strategy to insert the yeast gene into the bacterial plasmid. Below is a map of the area of the yeast genome surrounding the gene in which you are interested. yeast promoter Gene is normally transcribed this way S X K N E X N The distance between each tick mark placed on the line above is 100 bases in length. Below are the enzymes you can use, with their specific cut sites shown 5’-XXXXXX-3’ 3’-XXXXXX-5’ This is the map of the plasmid. Bacterial promoter The plasmid is 5,000 bases long and the two farthest restriction enzyme sites are 200 bases apart. The plasmid has an ampicillin resistance gene somewhere on the plasmid distal from the restriction cut sites. S K X E N Xba I: Nde I: Sal I: EcoR I: Xho I: Kpn I: TCTAGA CATATG GTCGAC GAATTC CTCGAG GGTACC AGATCT GTATAC CAGCTG CTTAAG GAGCTC CCATGGName:_KEY_________________________ 2 (a) Which single restriction enzyme is the best choice for you use to design a way to get the insert into the vector if you can only use one single enzyme? Keep in mind that your goal is to have the yeast gene be expressed in the bacterial cells. NdeI (N). NdeI and XbaI are the only two enzymes that can excise the gene from the genome, as they are the only two enzymes that cut on both sides of the gene. NdeI is the only enzyme that cuts the yeast gene outside of the ORF but downstream of the yeast promoter (which you don’t want as part of your insert because you want the gene to be expressed in bacteria, which means it must be under the control of a bacterial promoter). (b) You follow the cloning strategy you chose in part (a). You do the digestion of the insert and the vector and then ligate the two digestions together. You then transform the ligation into bacteria and select for ampicillin resistance. You get three colonies on your transformation plate. You isolate plasmid from each one and cut each plasmid with the enzyme XbaI. You then run your three digestions on an agarose gel and see the following patterns of bands. Describe what each plasmid actually was that was contained in each of the three colonies. colony 1 colony 2 colony 3 Colony 1’s plasmid = Vector with Yeast Gene in the Right Orientation 5000 nts 4000 nts 3000 nts 2000 nts 1000 nts 500 ntsName:_KEY_________________________ 3 S K X E N E N X S K X E N S K X E N X N E When the yeast gene is ligated into the plasmid, there are 2 sites where XbaI can cleave (once in the plasmid and once after the end of the open reading frame of the yeast gene). This produces 2 bands, one is ~1250bp and the other is 5000bp. Colony 2’s plasmid = Vector Alone (religated to itself) When there is no yeast gene ligated into the plasmid, there is only one site that XbaI cuts, which produces linearized plasmid that migrates at 5000bp. Colony 3’s plasmid = Vector with Yeast Gene in the Wrong Orientation When the insert is ligated into the plasmid in the wrong orientation, XbaI again can cut at 2 sites, one in the plasmid and one at the end of the open reading frame of the yeast gene. But now the 2 XbaI sites are closer together and give 2 bands, one that is 5850bp and the other that is ~400bp. (c) Which colony’s plasmid do you actually want to use for your studies? You want colony #1 because you want the bacterial promoter to drive transcription of the yeast gene in the correct orientation, so that the correct strand of DNA is used as a template in transcription. (d) Which two restriction enzymes would you use to design a way to get the insert into the vector if you had to use two enzymes simultaneously?Name:_KEY_________________________ 4 K (KpnI) and X (XbaI) These are the only 2 enzymes that, after cutting, would exclude the yeast promoter, include the bacterial promoter and allow for the yeast gene to be ligated in the correct orientiation following the bacterial promoter. The only two other enzymes that would cut simultaneously to give the open reading frame flanked by two different enzyme sites are X and N, and this would lead to inserting the gene in backwards into the vector. (e) You transform your ligation planned in part (d) into bacteria and plate the bacteria on Petri plates containing ampicillin. (You actually transform six different ligation mixtures, which are described below, into six different populations of cells, and plate each transformation onto a different plate, because you want to do all of the correct controls.) The next day you come in to lab to look at how many colonies of bacteria are on each plate. You are really excited, because the number of colonies you see on each plate tells you that the entire procedure worked! Which of the three following patterns of number of colonies did you see in order to conclude that you had a successful transformation? Circle the correct pattern. In this table, DV = digested vector. DYG = digested yeast genome. Ligation Used Pattern 1 Pattern 2 Pattern 3 DV only + Ligase 200 0 0 DYG only + Ligase 0 200 0 Water + Ligase 0 0 0 DV + DYG + Ligase 200 200
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