MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. Claudette Gardeliii) Would Bam HIbe useful cloning? Why or why not? No. There’s only one Bam site in smack inthe middle of YFG. iv) Would XbaI be useful for cloning? Why or why not?No. There’s an Xba I site in the middle of the Ampicillin resistance gene. v) Would XhoI be useful cloning? Why or why not? No. There’s a Xho I site in the Ori of pNEW. You digest both plasmids with the appropriate restriction enzyme(s) and combine digests in a single tube and add DNA Ligase. You transform appropriate bacteria with this ligation mix. b) How would you identify transformants that have taken up pNew but not pOld-YFG? (Hint: 2 steps) Select transformants on Ampicillin plates. Replica plate colonies onto tetracycline medium to screen AmpR colonies for tetracycline sensitivity. Now that you have colonies that have taken up pNew, you realize that the YFGgene could have inserted in either orientation. You take 10 transformants, isolate plasmid DNA from them, and perform restriction digestion using Bam H1. You get the following results after running the digests out on an agarose gel. c) Fill in the electrode charges in the circles depicting how the gel was run. BAM H1 – DIGESTED PLASMIDS MW 1234 5678910(kb) -10 9 4 3 + d) Which plasmids have no inserts? 1 2 7 9 10 e) Which plasmids have YFG inserted? 3 4 5 6 8 f) Of the plasmids in e) which colonies have YFGinserted in a way that it will be expressed off Plac? 5 6 g) How could you have avoided choosing colonies harboring plasmids without inserts? How could you have screened the transformants for ones that had plasmids with the YFG gene? Plate the transformation mix onto Ampicillin X-gal plates. Pick white colonies. 2Question 2 You want to study protein targeting in yeast, but first you need to construct a strain that will help you with your research. Your goal is to clone the gene encoding the Fructose Receptor (FruR), a plasma membrane protein, and fuse it with a gene encoding Green Fluorescent Protein (GFP). a) You first make a cDNA library from yeast that is able to grow fructose. This is done by using the enzyme Reverse Transcriptase (RT) to make a DNA copy of each of the messenger RNA molecules in this yeast strain. RT, like other DNA polymerases, needs a primer to initiate elongation. You recall from earlier lectures that eukaryotic mRNA has a cap at the 5' end and is polyadenylated (An) at the 3' end. 5’CAP------------------------------------------------------3’polyA tail i) Which end of an RNA molecule (5' or 3') would you use as a template to design a primer? Circle one. 3' ii) Given what you know, as stated above, what would be a good 'universal' primer for an RT reaction? (Assume 10 nucleotides long is sufficient.) TTTTTTTTTT b) Using the primer you just designed you’ve synthesized millions of cDNA molecules but only a few encode the fructose receptor gene. i) First you need to clone these cDNAs into vectors. What needs to be upstream of your cDNA in the vector to insure strong expression of the fructose receptor? Promoter ii) You consider expressing the protein in bacteria. Why would this plan make it critical to have made a cDNA library rather than making genomic DNA library? Bacteria are unable to splice out the introns that would occur in a genomic sequence from eukaryotic DNA iii) You decide to transfrom your library into yeast. How would you isolate the plasmid clone expressing the fructose receptor? What yeast strain would you transform into? Wild-type Yeast Fructose Receptor Mutant Yeast iv) What medium could you use to plate the transformants that would select for the correct plasmid clone. Minimal Fructose medium (Fructose as the only carbon source.) 3c) Now that you have the FruRgene cloned, you want to amplify the gfpgene encoding Green Fluorescent Protein from some jellyfish cells. Your first step is to design primers so that you can use polymerase chain reaction (PCR) to amplify the region. Below is highlighted the sequence at each outside end of gfp. 5'...ACGTAAACGGCCACAAGTTCAGCGT.......ACCCCGACCACATGAAGCAGCACGACT...3' ||||||||||||||||||||||||| (800) ||||||||||||||||||||||||||| 3'...TGCATTTGCCGGTGTTCAAGTCGCA.......TGGGGCTGGTGTACTTCGTCGTGCTGA...5' i) Of the potential primers listed below, which 2 can you use to amplify the DNA of interest? (Circle the correct 2.) A: 5'-GTAAACGGCCACAAG-3' B: 5'-CATTTGCCGGTGTTC-3' C: 5'-CTTGTGGCCGTTTAC-3' D: 5'-AATGGCATATGCCGT-3' E: 5'-ATGAAGCAGCACGAC-3' F: 5'-GTCGTGCTGCTTCAT-3' G: 5'-CAGCACGACGAAGTA-3' ii) Schematically, using lines as DNA and filled in boxes and open boxes as primers, draw the products of the reaction starting from the double stranded piece of DNA below after it has gone through each of 2 rounds of replication. The binding sites of the primers are shown on the template below. 5' 3' 3' 5' + 5' 3' Primer1 (in excess) 5' 3' Primer2 (in excess) After 1 round of PCR After 2 rounds of PCR iii) Using PCR, you successfully amplified a piece of DNA. To confirm that it is indeed gfpyou sequence it. What is the sequence of the DNA on the gel below? (Be sure to label the 5' and 3' ends.) dATP dCTP dGTP dTTP 5'-AGCTGTATAGGTTGTC-3’ 4Question 3 You are pleased to discover that this is indeed a sequence ofgfp. Your goal is to make a fusion protein where the N-terminus of Green Fluorescent Protein is replaced by Fructose Receptor. Green Fluorescent Protein (GFP) is a protein isolated from jellyfish that fluoresces green when exposed to blue light (It has been demonstrated that some proteins do not require their extreme C terminus or their N terminus to function.) In this case, the first half of the fusion protein (the NH3 end) will be a fully functional Fru R protein, and the second half (the COOH end) will be a fully functional GFP protein. GFPN C Transmembrane domain Fructose Receptor Signal Sequence The sequence for the extreme 3' end of fruRand the 5' start of gfpis shown below. For both, the underlined indicates an in frame codon. For gfp, it corresponds to the start codon. 3’ end of fruR 5’...CTTAAGGCCTAGGTACC...3’ ||||||||||||||||| 3’...GAATTCCGGATCCATGG...5’ 5’ end of gfp 5’...AGGCCTTAAGCCTAGGCTAGCAATGGTACC...3’ |||||||||||||||||||||||||||||| 3’...TCCGGAATTCGGATCCGATCGTTACCATGG...5’ AflII: AvrII StuI NheI KpnI C^TTAAG C^CTAGG AGG^CCT G^CTAGC GGTAC^C GAATT^C GGATC^C TCC^GGA CGATC^G
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