Assignment Project in Chemical Biology, TFKE39 You have been given the task of isolating any of these 10 DNA sequences coding for a protein. The gene is isolated from a cDNA library and cloned into an expression vector for expressing the protein in E. coli. Your task will be to make a laboratory protocols in which you describe: Which protein does the DNA sequence codes for? Indicate how you came to this conclusion, which database did you use? Designing primers for amplification of the gene with the help of PCR Designing primers to amplify the gene based on a cDNA library, note 5 'or 3' end and the forward and reverse primers. Specify how it binds to DNA (eg mark with italics or bold) Justify your choice of primer construction and how you chose the PCR cycle to amplify the gene. Isolation of PCR fragments Specify how you purify the PCR fragment after amplification. Choice of vectors for expression in E. coli Justify your choice of vector, based on the information you received about the protein's properties (eg, large or small protein?, Membrane protein or cytosolic? Post translationally modified?) Strategy for cloning the gene into the selected vector How should the gene be cloned into the selected vector? Which restriction enzymes should be used? How should the gene be expressed? Under what conditions it should be done (culture temperature, antibiotic resistance, and induction, with what? The chance that the protein is able to express in E. coli Based on the properties of the protein (eg, large or small protein? Membrane protein or cytosolic? Post translational modified?) Speculate on the chance that your design will be possible to express in E. coli Describe methods how to characterize the protein Based on the protein function make suggestions about how to characterize the protein (eg, the protein enzyme activity? Is it possible to measure the protein stability with a spectroscopic method? The assignment will be sent to lgmar@ifm.liu.se at latest sunday 28th of October. NOTE: Submitted report should be in PDF format and in the format Assignment_tfke39_NAME.PDF Late assignments without justification will not be read. Lasse Martensson IFM-Chemistry lgmar@ifm.liu.se Assessment: Individual Project Plan 5-10 pages that are considered according to the attached evaluation grid with scale (U, 3,4,5) References Literature: • TA Brown, Gene Cloning and DNA Analysis, An Introduction Databases and molecular biology companies: • Expasy Proteomics Server http://www.expasy.ch/ Protein Data Bank (3d structures of proteins) http://www.rcsb.org • NEB Cutter On-line program to check the restriction sites http://tools.neb.com/NEBcutter2/index.php Companies that sell molecular biology products or services • Stratagene (including the QuikChange site-specific mutagenesis) http://www.stratagene.com • New England Biolabs (NEB) (among others restriction enzymes) http://www.neb.com • Qiagen (including products for the purification of DNA, plasmidprep kit) http://www.qiagen.com • Novagen (including vectors for expression of proteins) http://www.novagen.com • GATC (including performing DNA sequencing http://www.gatc.de Choose one (1) of the following DNA sequence for cloning and expression in E.coli A atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggt gatgttaatgggcacaaattctctgtcagtggagagggtgaaggtgatgcaacatacgga aaacttacccttaaatttatttgcactactggaaagctacctgttccatggccaacactt gtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacag catgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttac aaagatgacgggaactacaaatcacgtgctgaagtcaagtttgaaggtgataccctcgtt aatagaattgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaa atggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatgga atcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagac cattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattac ctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatcctt cttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataa B atggcttctcatcgtctgctcctcctctgccttgctggactggtatttgtgtctgaggct ggccctacgggcaccggtgaatccaagtgtcctctgatggtcaaagttctagatgctgtc cgaggcagtcctgccatcaatgtggccgtgcatgtgttcagaaaggctgctgatgacacc tgggagccatttgcctctgggaaaaccagtgagtctggagagctgcatgggctcacaact gaggaggaatttgtagaagggatatacaaagtggaaatagacaccaaatcttactggaag gcacttggcatctccccattccatgagcatgcagaggtggtattcacagccaacgactcc ggcccccgccgctacaccattgccgccctgctgagcccctactcctattccaccacggct gtcgtcaccaatcccaaggaatga C atgaactaccccgtgaatcccgacctgatgcccgcgctgatggcggtcttccagcatgtg cggacgcgcatccagagcgagctcgattgccagcgactcgacctgaccccgcccgacgtc catgtattgaagcttatcgacgaacaacgcgggctgaacctgcaggacctgggacgccag atgtgccgcgacaaggcactgatcacccggaagatccgcgagctggagggaagaaacctg gtccgccgcgagcgcaaccccagcgaccagcgcagcttccagctcttcctcaccgacgag gggctggccatccaccagcatgcggaggccatcatgtcacgcgtgcatgacgagttgttt gccccgctcaccccggtggaacaggccaccctggtgcatctcctcgaccagtgcctggcc gcgcaaccgcttgaggatatttaa D atgccccggcgcagcctgcacgcggcggccgtgctcctgctggtgatcttaaaggaacag ccttccagcccggccccagtgaacggttccaagtggacttattttggtcctgatggggag aatagctggtccaagaagtacccgtcgtgtgggggcctgctgcagtcccccatagacctg cacagtgacatcctccagtatgacgccagcctcacgcccctcgagttccaaggctacaat ctgtctgccaacaagcagtttctcctgaccaacaatggccattcagtgaagctgaacctg ccctcggacatgcacatccagggcctccagtctcgctacagtgccacgcagctgcacctg cactgggggaacccgaatgacccgcacggctctgagcacaccgtcagcggacagcacttc gccgccgagctgcacattgtccattataactcagacctttatcctgacgccagcactgcc agcaacaagtcagaaggcctcgctgtcctggctgttctcattgagatgggctccttcaat ccgtcctatgacaagatcttcagtcaccttcaacatgtaaagtacaaaggccaggaagca ttcgtcccgggattcaacattgaagagctgcttccggagaggaccgctgaatattaccgc taccgggggtccctgaccacacccccttgcaaccccactgtgctctggacagttttccga aaccccgtgcaaatttcccaggagcagctgctggctttggagacagccctgtactgcaca cacatggacgacccttcccccagagaaatgatcaacaacttccggcaggtccagaagttc gatgagaggctggtatacacctccttctcccaagtgcaagtctgtactgcggcaggactg agtctgggcatcatcctctcactggccctggctggcattcttggcatctgtattgtggtg gtggtgtccatttggcttttcagaaggaagagtatcaaaaaaggtgataacaagggagtc atttacaagccagccaccaagatggagactgaggcccacgcttga E atggatggtacaagaacttcacttgacattgaagagtactcggatactgaggtacagaaa aaccaagtactaactctggaagaatggcaagacaagtgggtgaacggcaagactgctttt catcaggaacaaggacatcagctattaaagaagcatttagatactttccttaaaggcaag agtggactgagggtattttttcctctttgcggaaaagcggttgagatgaaatggtttgca gaccggggacacagtgtagttggtgtggaaatcagtgaacttgggatacaagaatttttt acagagcagaatctttcttactcagaagaaccaatcaccgaaattcctggaaccaaagta tttaagagttcttcggggaacatttcattgtactgttgcagtatttttgatcttcccagg acaaatattggcaaatttgacatgatttgggatagaggagcattagttgccattaatcca ggtgatcgcaaatgctatgcagatacaatgttttccctcctgggaaagaagtttcagtat ctcctgtgtgttctttcttatgatccaactaaacatccaggtccaccattttatgttcca catgctgaaattgaaaggttgtttggtaaaatatgcaatatacgttgtcttgagaaggtt gatgcttttgaagaacgacataaaagttggggaattgactgtctttttgaaaagttatat ctacttacagaaaagtaa F atgagctcccagattcgtcagaattattccaccgacgtggaggcagccgtcaacagcctg gtcaatttgtacctgcaggcctcctacacctacctctctctgggcttctatttcgaccgc gatgatgtggctctggaaggcgtgagccacttcttccgcgaactggccgaggagaagcgc gagggctacgagcgtctcctgaagatgcaaaaccagcgtggcggccgcgctctcttccag gacatcaagaagccagctgaagatgagtggggtaaaaccccagacgccatgaaagctgcc atggccctggagaaaaagctgaaccaggcccttttggatcttcatgccctgggttctgcc cgcacggacccccatctctgtgacttcctggagactcacttcctagatgaggaagtgaag cttatcaagaagatgggtgaccacctgaccaacctccacaggctgggtggcccggaggct gggctgggcgagtatctcttcgaaaggctcactctcaagcacgactaa G atgacagcagacaaattggttttctttgtgaatggcagaaaggtggtggagaaaaatgca gatccagagacaacccttttggcctacctgagaagaaagttggggctgagtggaaccaag ctcggctgtggagaggggggctgcggggcttgcacagtgatgctctccaagtatgatcgt ctgcagaacaagatcgtccacttttctgccaatgcctgcctggcccccatctgctccttg caccatgttgcagtgacaactgtggaaggaataggaagcaccaagacgaggctgcatcct gtgcaggagagaattgccaaaagccacggctcccagtgcgggttctgcacccctggcatc gtcatgagtatgtacacactgctccggaatcagcccgagcccaccatggaggagattgag aatgccttccaaggaaatctgtgccgctgcacaggctacagacccatcctccagggcttc cggacctttgccagggatggtggatgctgtggaggagatgggaataatccaaattgctgc atgaaccagaagaaagaccactcagtcagcctctcgccatctttattcaaaccagaggag ttcacgcccctggatccaacccaggagcccatttttcccccagagttgctgaggctgaaa gacactcctcggaagcagctgcgatttgaaggggagcgtgtgacgtggatacaggcctca accctcaaggagctgctggacctcaaggctcagcaccctgacgccaagctggtcgtgggg aacacggagattggcattgagatgaagttcaagaatatgctgtttcctatgattgtctgc ccagcctggatccctgagctgaattcggtagaacatggacccgacggtatctcctttgga gctgcttgccccctgagcattgtggaaaaaaccctggtggatgctgttgctaagcttcct gcccaaaagacagaggtgttcagaggggtcctggagcagctgcgctggtttgctgggaag caagtcaagtctgtggcgtccgttggagggaacatcatcactgccagccccatctccgac ctcaaccccgtgttcatggccagtggggccaagctgacacttgtgtccagaggcaccagg agaactgtccagatggaccacaccttcttccctggctacagaaagaccctgctgagcccg gaggagatactgctctccatagagatcccctacagcagggagggggagtatttctcagca ttcaagcaggcctcccggagagaagatgacattgccaaggtaaccagtggcatgagagtt ttattcaagccaggaaccacagaggtacaggagctggccctttgctatggtggaatggcc aacagaaccatctcagccctcaagaccactcagaggcagctttccaagctctggaaggag gagctgctgcaggacgtgtgtgcaggactggcagaggagctgcatctgcctcccgatgcc cctggtggcatggtggacttccggtgcaccctcaccctcagcttcttcttcaagttctac ctgacagtccttcagaagctgggccaagagaacctggaagacaagtgtggtaaactggac cccactttcgccagtgcaactttactgtttcagaaagaccccccagccgatgtccagctc ttccaagaggtgcccaagggtcagtctgaggaggacatggtgggccggcccctgccccac ctggcagcggacatgcaggcctctggtgaggccgtgtactgtgacgacattcctcgctac gagaatgagctgtctctccggctggtcaccagcacccgggcccacgccaagatcaagtcc atagatacatcagaagctaagaaggttccagggtttgtttgtttcatttccgctgatgat gttcctgggagtaacataactggaatttgtaatgatgagacagtctttgcgaaggataag gttacttgtgttgggcatatcattggtgctgtggttgctgacaccccggaacacacacag agagctgcccaaggggtgaaaatcacctatgaagaactaccagccattatcacaattgag gatgctataaagaacaactccttttatggacctgagctgaagatcgagaaaggggaccta aagaaggggttttccgaagcagataatgttgtgtcaggggagatatacatcggtggccaa gagcacttctacctggagactcactgcaccattgctgttccaaaaggcgaggcaggggag atggagctctttgtgtctacacagaacaccatgaagacccagagctttgttgcaaaaatg ttgggggttccagcaaaccggattgtggttcgagtgaagagaatgggaggaggctttgga ggcaaggagacccggagcactgtggtgtccacggcagtggccctggctgcatataagacc ggccgccctgtgcgatgcatgctggaccgtgatgaggacatgctgataactggtggcaga catcccttcctggccagatacaaggttggcttcatgaagactgggacagttgtggctctt gaggtggaccacttcagcaatgtggggaacacccaggatctctctcagagtattatggaa cgagctttattccacatggacaactgctataaaatccccaacatccggggcactgggcgg ctgtgcaaaaccaaccttccctccaacacggccttccggggctttggggggccccagggg atgctcattgccgagtgctggatgagtgaagttgcagtgacctgtgggatgcctgcagag gaggtgcggagaaaaaacctgtacaaagaaggggacctgacacacttcaaccagaagctt gagggtttcaccttgcccagatgctgggaagaatgcctagcaagctctcagtatcatgct cggaagagtgaggttgacaagttcaacaaggagaattgttggaaaaagagaggattgtgc ataattcccaccaagtttggaataagcttcacagttccttttctgaatcaggcaggagcc ctacttcatgtgtacacagatggctctgtgctgctgacccacggggggactgagatgggc caaggccttcataccaaaatggtccaggtggccagtagagctctgaaaatccccacctct aagatttatatcagcgagacaagcactaacactgtgcccaacacctctcccacggctgcc tctgtcagcgctgacctcaatggacaggccgtctatgcggcttgtcagaccatcttgaaa aggctggaaccctacaagaagaagaatcccagtggctcctgggaagactgggtcacagct gcctacatggacacagtgagcttgtctgccactgggttttatagaacacccaatctgggc tacagctttgagactaactcagggaaccccttccactacttcagctatggggtggcttgc tctgaagtagaaatcgactgcctaacaggagatcataagaacctccgcacagatattgtc atggatgttggctccagtctaaaccctgccattgatattggacaggtggaaggggcattt gtccagggccttggcctcttcaccctagaggagctacactattcccccgaggggagcctg cacacccgtggccctagcacctacaagatcccggcatttggcagcatccccattgagttc agggtgtccctgctccgcgactgccccaacaagaaggccatctatgcatcgaaggctgtt ggagagccgcccctcttcctggctgcttctatcttctttgccatcaaagatgccatccgt gcagctcgagctcagcacacaggtaataacgtgaaggaactcttccggctagacagccct gccaccccggagaagatccgcaatgcctgcgtggacaagttcaccaccctgtgtgtcact ggtgtcccagaaaactgcaaaccctggtctgtgagggtctaa H atggtgaaggtcggtgtcaacggatttggccgtattggccgcctggtcaccagggctgcc ttctcttgtgacaaagtggacattgttgccatcaacgaccccttcattgacctcaactac atggtctacatgttccagtatgattctacccacggcaagttcaatggcacagtcaaggct gagaatgggaagctggtcatcaatgggaaacccatcaccatcttccaggagcgcgatccc gctaacatcaaatggggtgatgctggtgctgagtatgtcgtggagtctactggcgtcttc accaccatggagaaggctggggctcacctgaagggtggtgccaaaagggtcatcatctcc gccccttccgctgatgcccccatgtttgtgatgggtgtgaaccacgagaaatatgacaac tccctcaagattgtcagcaatgcatcctgcaccaccaactgcttagcccccctggccaag gtcatccatgacaactttggcatcgtggaagggctcatgaccacagtccatgccatcact gccactcagaagactgtggatggcccctctggaaagctgtggcgtgatggccgtggggca gcccagaacatcatccctgcatccactggtgctgccaaggctgtgggcaaggtcatccca gagctgaacgggaagctcactggcatggccttccgtgttcctacccccaatgtatccgtt gtggatctgacatgccgcctggagaaacctgccaagtatgatgacatcaagaaggtggtg aagcaggcggccgagggcccactaaagggcatcctgggctacactgaggaccaggttgtc tcctgtgacttcaacagcaactcccattcctccacctttgatgctggggctggcattgct ctcaatgacaactttgtgaagctcatttcctggtatgacaatgaatatggctacagcaac agggtggtggacctcatggcctacatggcctccaaggagtaa I atgttccgtttgccagtttcgcttgctcgctcctccattagccgccagttggccatgcgc ggctatgccaaggatgtgcgtttcggacccgaggtgcgcgccatgatgctccagggcgtc gatgtgctggccgatgctgtggcagtgaccatgggccccaagggtcgcaatgtgatcatt gagcagtcgtggggctcgcccaaaatcaccaaggacggcgtcactgtggccaagtccatt gagctgaaggacaagttccagaacatcggtgccaagctggtccaggatgtggccaacaat accaacgaggaggccggtgatggcaccaccacggccaccgttctggctcgcgccattgcc aaggagggcttcgagaagatctccaagggcgccaatcccgtcgagattcgtcgcggtgtc atgctggccgttgagaccgtcaaggacaaccttaagaccatgtcgcgccccgttagcacg cccgaggagatcgcccaggtggccaccatctcggccaacggtgatcaggccatcggcaat ctcatcagcgaggccatgaagaaggtgggacgcgatggcgttatcaccgtcaaggatggc aagaccctcaccgatgagttggaggttatcgagggcatgaagttcgatcgcggctacatc tcgccgtacttcatcaactcctcgaagggcgccaaggtggagttccaagatgccctcctc ctgctgtccgagaagaagatctcatcggtgcagagcatcattcccgctctggagctggcc aagtcgcagcgcaagccgctggtcatcattgccgaggatatcgatggtgaggccctgagc accctggtggtgaaccgcctcaagatcggccttcaggtggctgccgtcaaggcacccggc ttcggcgacaaccgcaagagcaccctcaccgatatggccatcgcctccggcggcattgtc ttcggcgatgatgctgatctcgtcaagctggaggacgtcaaggttagcgatttgggccag gtcggcgaggtggtgatcaccaaggacgacaccctgctgctgaagggcaagggcaagaag gacgatgtgctccgtcgcgccaatcagataaggaccaagatcgaggacaccacctccgag tacgagaaggagaagctgcaggagcgtctggcccgcttggcctccggcgttgccctgctg cgcgttggcggctccagcgaggtggaggtcaacgagaaggaggatcgcgtccacgatgcc ctgaacgccacccgtgctgccgtcgaagagggaatcgttcccggaggcggcacccgtctc gtccggttgattgagaagctcgagggtgtcgagaccaccaacgaggatcagaaactcggc gtggagattgtgcgtcgtgcctcgcgaatgccatgcatgacgattgccaagaacgccggc gttgatggcgccatggtggtggccaaggtggagaatcaggccggtgactacggctacgat gcgctgaagggcgaatacggcaacctgatcgagaagggcatcatcgatcccaccaaggtg gtgcgcaccgccatcacagacgcctccggtgtggcctcactgctgaccaccgccgaggct gtcgtcaccgagatccccaaggaggatggcgctcctgccatgcccggcatgggtggtatg ggcggcatgggaggcatgggcggcatgggtggcatgatgtaa J atggctatgataccttcaatttcaaagttgctttttgttgcaatatgtctttttgtttat atgggtttgtcatttggtgatttttctattgtgggttattctcaaaatgacttgacatcc actgaaagacttattcagctatttgaatcgtggatgttgaagcacaataagatttataag aatattgatgaaaaaatctacagatttgaaatttttaaagataatcttaaatatattgat gagacaaataagaaaaataacagttattggcttggattaaatgtgtttgctgatatgagc aatgatgaattcaaagaaaagtatactggttctattgctggaaattatacaacaaccgaa ctatcatacgaagaagtgcttaatgatggtgatgtaaatatcccggagtatgtcgattgg agacaaaaaggagctgtcactcctgtaaaaaatcagggttcttgtggtagttgttgggca ttctcagctgttgtaactatagagggaataattaagattagaactgggaacttaaatgaa tactcagagcaagaactgcttgactgcgacagacgtagctacgggtgcaatggaggttac ccttggagtgcacttcaattagtggctcaatatggtattcactacagaaatacttaccca tatgagggagtgcaacgttattgtcgctcaagggagaaaggtccttatgcagccaaaacc gatggggttcgacaagtgcaaccatataatgaaggggctctcttatattcaattgcaaac caacctgtgagcgttgtccttgaagctgctggaaaagatttccaattatatagaggggga atatttgtggggccatgcggaaacaaagtagatcatgcagtcgcagcagttgggtatgga ccaaattacatactcataaagaattcatggggtacaggatggggtgaaaatggatatata agaatcaaaagaggcactggaaactcctatggagtatgtggactttatacaagctcattc tatcctgttaaaaactga Evaluation template for assignment in the project course in Chemical Biology Assignment Project Chemical Biology Student: Student: Basic criteria 1 2 Purpose and content criteria Average score: Criteria includes (Max:28 points) 2 3 4 1 2 3 4 The assignment linguistically and graphically consistent. Adequate factually deep, moderately technical language Criteria includes 3 Clear and well-motivated Specific Objectives 4 Adequate background summary 5 Sophisticated methodological content in terms of both experimental and practical aspects 6 Feasibility - overall assessment based on the application 7 originality, innovation, creativity! Feedback: 1