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Download 1 Biology 437 Fall 2015 Syllabus Biology 437: LABORATORY ON
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Biology 437 Fall 2015 Biology 437: LABORATORY ON DNA MANIPULATION Instructor: Professor Robert Kranz ( [email protected] ) Syllabus TAs: Jared Andrews ([email protected] ) Nicole Fazio ( [email protected] ) Mary Mathyer ( [email protected] ) Sarah Santiago ( [email protected] ) Rebstock room 126: Wed & Fri 2:30-6pm; Thursday 4-5pm Textbook: none ("Fermentas" catalog with technical information is used). I. You will be given the following handouts on the first day of class: 1. Student Questionnaire 2. Bio 437 course overview and goals 3. Class Syllabus (timing subject to change throughout semester) 4. Guidelines for keeping a Laboratory Notebook 5. Guide for the lab write-up 6. Laboratory Safety Rules 7. Handouts for this week’s lab (Lab I) 8. A lab notebook and 3-ring binder II. How is the class graded? • Two in-term exams: 1st is 100 pts; 2nd is 50 pts • Two Problem sets: 1st is 50 pts; 2nd is 25 pts • Subjective 25 pts given after the course is over, decided by all instructors (eg. missed or late labs, assignments, sloppy work) • Lab Write-up (Paper): 100 pts • Lab Notebook 50 points (collected twice, 25 pts each time) Total 400 points III. Office Hours: (Dr. Kranz and/or at least two TAs will be at all labs and lectures so these 8 hours per week will have ample time for discussion, or alternatively, email Dr. Kranz) IV. Bring your lab notebook and binder to every period. V. Lectures will be interspersed into lab experiments where they fit. VI. Attendance is mandatory. Be on time because a short lecture outlining the day’s procedure will take place at the start of some labs. VII. There is NO CLASS on Friday of Fall Break (Oct16). There are no classes the week of Thanksgiving Break (Nov 25-29). Last day of classes is Friday, Dec 4. 1 Week (and W,Th, Friday Dates) Lecture(s) Experiments Important info & dates 1 Lect 1&2 Intro, DNA Calibrate pipettes/sterile technique Cloning assignments will be given. Individual meeting about cloning projects. Primer design. Set-up E. coli overnights to prep DNA for cloning projects. Preparation of DNA for cloning projects. Diagnostic digest of DNA & DNA agarose gel. Prepare oligonucleotides (primers) for Quikchange PCR reaction for sitedirected mutagenesis. DpnI digest. Transformation of reactions into E. coli. Count colonies from transformation. Set-up overnights to prep clone DNA. Module 1 starts (Aug 26,27, 28) 2 (Sept 2,3,4) 3 (Sept 9,10,11) 4 (Sept 16,17,18) 5 (Sept 23,24,25) 6 Lect 3&4 PCR, molecular cloning, sitedirected mutagenesis Lect 5&6 Biology behind module 1 projects Prepare clone DNA. Diagnostic digest and DNA agarose gel. Prepare sequencing reaction. Lect 7 DNA sequencing Analyze sequencing reactions. Set-up overnights to freeze strains or if necessary, troubleshoot cloning. Lect 8 Homologous recombinatio n/knockouts Trouble-shooting, discussion of next experiments. Lect 9 Impact of (Sept 30, Oct Genome 1, 2) sequencing Trouble-shooting, discussion of next experiments. Wed: lecture, pipetting,primer design Thur: Sterile technique, start E.coli cultures, finish lecture Fri: DNA minipreps and analyses Problem Set 1 handed out (due week 6, Wed, Sept 30, 2:30pm) Problem Set 1 due week 6, Wed, Sept 30, 2:30pm) 2 7 Lect 10 RNA and RNA methods Trouble-shooting, discussion of next experiments. Protein work on mutated genes (proteins), if feasible Lect 11 RNA and RNA methods Trouble-shooting, discussion of next experiments. Protein work if feasible Exam 1 on Wed, Oct 14 (covers module 1 Continue protein work. Module 2 starts For Module 2, set up corn seeds for roots vs shoots analysis, RNA studies. Module 1 will continue as time permits. 10 Finish module 1. (Oct 28,29,30) Module 2: Design primers for corn transgenes. Practice tissue prep and RNA purification Tissue prep and RNA purification. Problem Set 2 handed out (due week 12, Wed, Nov 11, 2:30pm (Oct 7,8,9) 8 (Oct 14,15, no class on Oct 16) 9 (Oct 21,22,23) Lect 12 Biology behind Module 2 projects 11 Make cDNA. (Nov 4,5,6) Perform RT-PCR to become familiar with the technique. 12 Lect 13 Other (Nov 11,12,13) technologies in nucleic acid manipulations 13 (Nov 18,19,20) (Dec 2,3,4) Fall Break on Friday, Oct 16) OCT 30 Halloween pumpkin carving Perform RT-PCR/QRTPCR. Analyze the results Problem Set 2 due Wed, Nov 11, 2:30pm Trouble-shooting, discussion of next experiments. Discuss which project to write up into paper. Thanksgiving (Nov 25-29) 14 and lectures 1 thru 9) Thanksgiving Lect 14 Summary of Bio437 successes, writing a scientific paper Wrap-up final experiments with Module 1&2 Exam 2 on Wed, Dec 2—covers module 2 and lectures 9 thru 13 Clean up lab /DipN Dots & pizza party 3 Bio 437 (Fall, 2015): General Overview and two lab modules: from Prof Kranz The magnificent boom in biotechnology since the 1970s is a direct result of the ability to manipulate and measure nucleic acids. These advancements have revolutionized research in health and the environment. This course is designed as a hands-on experience that will provide the student with an understanding of how research on nucleic acids is performed and that also may be of use in the biotechnology field. Module 1 is based on DNA technologies while module 2 provides hands-on research in RNA methods. Module 1) Cloning genes, directed mutagenesis, and sequence analysis: Projects for Table A: Organisms use cytochrome c for respiration and photosynthesis. For example, eukaryotes have mitochondria whereby cellular respiration via electron transport is used to synthesize ATP and for other processes essential to life. All mitochondria possess cytochrome c (and cytochrome c1) for this electron transport. All cytochrome c proteins have heme that is covalently attached to the protein by two thioether bonds (at two cysteines in the protein, cys15-X-X-cys18-his19). This covalent attachment requires the mitochondrial protein called holocytochrome c synthase (HCCS). Since the gene for HCCS and other mitochondrial proteins are the targets of many human diseases and cytochrome c is also needed for programmed cell death (apoptosis), it is important to understand how HCCS functions, and to be able to control it. Very recently the Kranz lab was able to clone the human HCCS and the human cytochrome c and show it is functional in Escherichia coli (ie the heme is attached by HCCS). This makes it feasible to study HCCS and to potentially biosynthesize new types of cytochromes c. Bio437 students will engineer into the human cytochrome c (gene) new amino acids (substitutions or deletions in codons) that have never been made, using the genetically engineered E.coli to biosynthesize these novel cytochrome c. Projects for Tables B and C: Every organism on earth can use the 20 natural amino acids in their coding sequences--- this is at the core of the “central dogma” of biology. A relatively new technology has emerged whereby it is possible to “recode” a gene by genetic engineering, facilitating the use of nonstandard amino acids (NSAA). We can thus engineer anywhere in a protein this “new” NSAA by genetically changing the codon and employing engineered protein synthesis machinery that will recognize the new NSAA. For this class, we will genetically engineer (recode) genes that encode a cytochrome c from bacteria (called cytochrome c4). This will allow for overproduction, purification and characterization of cytochrome c proteins in Escherichia coli that now will incorporate the NSAA of our choice. Main scientific questions: Those students constructing alterations in the human cyt c will test whether the variants interact with HCCS and can be assembled with heme. For students “recoding” for NSAA, a particular NSAA (eg. Benzoylphenylalanine) crosslinks to any other residue near it, so will properly positioning this NSAA crosslink the cytochrome c to the cytochrome c synthetase that makes cytochrome c? This would define where interaction is between the two proteins. Will these recoded cytochrome c’s be functional? Will these engineered (recoded) cytochrome c proteins allow incorporation of fluorescent NSAAs, to make entirely new chromophores? We hope to demonstrate how use of mass spectrometry will prove that a NSAA has actually been incorporated into a cyt c, which has never been accomplished before. Training rationale: You will learn to design, clone, and analyze the sequence for the mutations. This process is called genetic engineering and is critical for basic science, biotechnology, and biomedical research. Through this project you will gain hands on skills in DNA manipulation that will be translatable to a wide array of research fields. We will pursue overexpression in E.coli of the new cytochromes you engineered and its analysis by spectroscopic and other techniques. TAs: Sarah Santiago, Mary Mathyer, and Nicole Fazio Module 2) RNA analyses: RT-PCR and q-PCR on mRNAs for gene expression. Students will carry out projects using a eukaryote (i.e. the corn plant Zea mays). RNAseq, q-PCR, and microarrays are now the gold standard technologies for comprehensive understanding of what genes are involved (i.e. expressed) under certain conditions (or diseases). In previous years, Bio 437 students carried out 4 microarray analyses and q-PCR of genes expressed in Arabidopsis (a weed) under nitrogen or phosphate stress (ie. fertilizer-deficient plants). This year we will analyze genetically modified organisms (GMO corn) that have engineered traits (“transgenes”) that farmers use to outcompete weeds and fight off insect pests. These GMO corn have been transformed with genes (transgenes) from bacteria, called “round-up ready, RR” or BT toxin genes. (The BT toxins (encoded by cry genes) kill insects, the RR gene is for resistance to a herbicide called Round-up.) The main scientific questions: address the levels and location of expression of BT (Cry) and RR genes in GMO corn. Training rationale for this project: Learning the details of mRNA analysis, including tissue preparation, RNA purification, cDNA synthesis, RT-PCR, q-PCR, global gene expression profiling. (eg. many studies on global gene expression profiling of human cancer cells are unraveling what genes are involved in the cancer state, and require these technologies for analyses). TA: Jared Andrews Other learning goals: Although we will not sequence or annotate an entire genome, we will emphasize throughout the course how and why whole genome sequences have revolutionized biology and biotechnology. A few lectures will cover other methods of biotechnology (nucleic acid manipulation) which we will not use (eg. RNAi, CRISPRs, gene knock-outs, Chromatin immunoprecipitation, RNAseq). 5