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PS_1_older_key - Biology 115 Winter 2005 January 14 Answers...

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Biology 115, Winter 2005 January 14 Answers to problem Set #1 1. You want to express the Purple gene in bacteria. This is a mouse gene responsible for bright purple streaks commonly observed in the hair of some mouse species. You have the cloned Purple gene, with its natural promoter, and also a bacterial expression vector. Restriction maps for both of these are shown below. (A restriction map is a cartoon/map showing the relative order and position ofrestriction sites in a piece of DNA) Describe a strategy for cloning the Purple gene into the expression vector. For the sake of simplicity, assume that DNA sequences that specify translation of the Purple gene are supplied on the fragment that you clone into the expression vector. (Note: this process of creating a recombinant from two cloned genes is often called subcloning ) Cut out the Xho I/Eco RI fragment containing the Purple gene from the first vector, and ligate it into Xho I/Eco RI digested expression vector. This is because: 1. The Purple gene should be left intact. Thus, Pst I is a bad choice of an enzyme to use. This leaves either Xho I or Bgl II on one side of Purple, and either Eco RI or Bam HI on the other side of Purple as possible choices. 2. The orientation of the Purple gene with respect to the promoter transcribing it must be preserved, otherwise the wrong strand of the Purple gene will be transcribed. Looking at the expression vector, this rules out using Bam HI, since this site is preceded by neither an Xho I nor a Bgl II site. Similarly, Bgl II cannot be used since, in the expression vector, the second Bgl II site is not followed by either an Eco RI or a Bam HI site. Thus, XhoI and Eco RI must be used to cut out the Purple gene and clone it into the expression vector in the correct orientation. 2. The recognition sequence for Not I is 5'-GCGGCCGC-3' 3'-CGCCGGCG-5' and recognition sequence for Eag I is 5'-CGGCCG-3'. 3'-GCCGGC-5' Both of these restriction enzymes cut their sites so as to leave 4 basepair 5' overhangs. Purple Bam HI Pst I Pst I Eco RI Bgl II Xho I Amp r ori Amp r ori Purple gene promoter Bacterial Promoter Bgl II Pst I Xho I Bam HI Eco RI Bgl II Eco RI
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a. Draw out both strands of the DNA sequences left by these enzymes after they have cut their sites. (for example, Bam HI recognizes 5’-G GATCC -3’ 3’-CCTAG G -5’ and leaves 4 basepair, 5’ overhangs. After cutting, Bam HI leaves two fragments, 5’-G GATCC -3’ 3’-CCTAG G -5’) Not I 5'-GC GGCCGC -3' 3'-CGCCGG CG -5' Eag I 5'-C GGCCG -3' 3'-GCCGG C -5' b. Can you ligate a DNA fragment cut with Not I into a vector cut with Eag I? If so, draw out the sequence of the junctions of the resulting recombinant DNA and explain how they differ from the parental DNA sequences they were derived from.
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