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Download Examination in Gene Technology, TFKE38 2011-10-18
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Examination in Gene Technology, TFKE38 2011-10-18 1) Explain briefly the following concepts in molecular biology a) Competent Cells b) Transfection c) Fagmid d) operons d) Cos-sites (10 p) 2) Plasmid pBR322 (Figure 1) was digested with restriction enzyme Pvu1. The digested plasmid was ligated with the Pvu1 digested gene coding for protein X and the mixture was transformed into E. coli cells. a) What antibiotics should be added to the medium (agarplate) to select cells that have incorporated the gene for proteinX? (2 p) b) After transformation colonies were obtained that were resistant to both Ampicillin and Tetracycline. Can you give a reasonable explanation for this? (2 p) c) To ligate the gene for protein X into pBR322, only one restriction site was used. What might this have for disadvantages? (2 p) d) To facilitate the ligation the plasmid was treated with the enzyme alkaline phosphatase. How does this enzyme work and how can it improve the ligation frequency. (4 p) Figure1 Schematic drawing of plasmid pBR322 3) You have obtained a human cDNA library and will try to clone the protein FABP, a protein that has a very conserved protein sequence among eukaryotes. a) What distinguishes a cDNA library from a genomic library? (2p) b) How do you proceed to create a cDNA library. Sketch a possible way to construct a cDNA library, indicating the necessary enzymes. (5p) c) Outline how you go about identifying the clone containing the gene for the protein FABP. (3 p) 4) You want to study the ribosomal proteins and has therefore chosen to clone protein L21. The figure below (figure 2) shows the DNA sequence and protein sequence of L21. NOTE: This question provides a total of 20 points a) Construct primers to find the gene in a cDNA library (2p) b) In order to quickly clone your desired fragments without the use of restriction digest instead you use the socalled TA cloning technique.. How does the TA cloning work? What are the requirements for the DNA polymerase used in this technique, how is the vector treated? (6p) c) For the transformation, you use two different controls, transformation and ligation control. What are the purposes of these controls and what are the expected results of those checks (2p) d) To study the protein spectroscopically you want by site-directed mutagenesis to replace Tyr7 to a tryptophan (Tyr7 (Y7 in one-letter code)) selected in the protein sequence in bold and underlined). Design primers for using the QuikChange method to do this amino acid replacement. Give a justification for how you design these primers. (Utilities Genetic code in Appendix) (4p) e) Describe how the QuikChange method works and what happens in the various stages. (6p) A 5´atgactaactccaagggttacagacgtggcacgagggacttgttctctcgcaaattccgtaaacatggaac cattcccttatccacgtacgtgaaaacgtacaaagtgggcgatattgttgatattaagggaaatggtgctg tacaaaagggtatgccgtataaagtttatcacggtaaaactggtcgcgtattcaacgtaactgcgcacgcc ctgggagtcatcgtgaacaagagagtacgtggacgtatcattgccaagaggatcaatgttcgcatcgagca tctgagccattcaaagtgtagggatgatttcctcaaacgcgtcaaagagaacgagagactcaggaaggagg caaaggagaaaaatgttcgcgttcagcttaaacgacaaccggccgaaccatctaaagcacatatcgtgtca ggacgtgaagcgccaatcttactcgcaccagttccctatgaatttattgcttaa-3´ B MTNSKGYRRG TRDLFSRKFR KHGTIPLSTY VKTYKVGDIV DIKGNGAVQK GMPYKVYHGK TGRVFNVTAH ALGVIVNKRV RGRIIAKRIN VRIEHLSHSK CRDDFLKRVK ENERLRKEAK EKNVRVQLKR QPAEPSKAHI VSGREAPILL APVPYEFIA Figure 2 DNA sequence (A) and amino acid sequence (B) of L21 protein Appendix Genetic code