BI425/525: Advanced Molecular Biology Research Lab, Fall 2017

Transcription

1 BI425/525: Advanced Molecular Biology Research Lab, Fall 2017 Instructor- Alice Barkan. Teaching Assistant-Carolyn Brewster This is an intensive advanced laboratory course that will involve students in research that combines genetic, molecular biology and genomic approaches to dissect a complex cellular process: the biogenesis of the chloroplast. Students will gain hands-on experience with common molecular biology techniques, bioinformatic tools and "next generation" DNA sequencing methods. Students will use their knowledge of genetics, gene expression, and photosynthesis acquired in introductory coursework, and they will build on that foundation by learning about transposable elements, nuclearorganellar interactions, gene families, genome evolution, and more. Lectures and assigned readings will describe the biological context of this research and the theoretical basis of the techniques employed. Student-directed discussion of research articles and current topics related to the course content will be incorporated throughout the term. The broad goals of the course are to: Provide research training as a stepping stone to research in a UO lab and/or post-graduation employment. Synergize with upper level courses that focus on primary research literature by providing hands-on experience with common experimental methodologies. Student Learning Outcomes. Students in this course will: Gain hands-on experience with the theory and practice of common methods in molecular biology (e.g. western blotting, DNA/RNA purification, PCR, RT-PCR, molecular cloning, recombinant protein expression, next-generation DNA sequencing). Be trained in the use of genome databases and tools for making inferences about gene structure and function. Learn basic laboratory skills such as how to keep a proper laboratory notebook, make solutions, etc. Learn about biology that is only minimally covered in other UO courses (e.g. transposable elements, the biogenesis of chloroplasts and mitochondria, plant biotechnology). Develop the critical thinking and creative skills associated with data evaluation, hypothesis formulation, and experimental design. Grades will be based on the following components: Lab notebook checks (three graded checks; 15% of grade: Is the goal of the experiment clearly stated? Are the procedures outlined in sufficient detail? Is the data properly labeled? Are the conclusions summarized? Are implications and suggestions for future experiments noted? Quizzes (Four quizzes, 28% of grade) "Journal club" presentation (15%). Students will work in small groups to present a research article or overview of current topic related to the course content. See syllabus for topic choices. Final Symposium Talk (15%): Grades will be based on clarity of presentation, understanding of the material, ability to answer questions clearly and correctly. Final Paper (20%)- This project summary should include the question posed, experimental approach, and conclusions at each step, the final conclusions and the reasoning that led to them. Describe new questions that arise from your results and where you would go from here if you were to continue the project. Discretionary points (7%)- Are equipment and reagents used properly? Is the student cooperative? Does the student participate actively in discussions and display independent thought? 1

2 BI425/525 Fall 2017 Overview of Experimental Goals Two independent projects will be interwoven during the term: Goal #1: Identify the gene whose disruption causes a specific non-photosynthetic mutant phenotype in maize - analyze mutant phenotypes by Western blot analysis of photosynthetic complexes - identify candidate causal mutations: Mu-Illumina analysis to find Mu insertions that cosegregate with this phenotype - prioritize candidates for followup: Bioinformatic analyses of genes disrupted by cosegregating Mu insertions - test best candidates by PCR to evaluate genetic linkage to the mutant phenotype - quantify mrna from best candidate gene by RT-PCR to determine the degree to which the transposon insertion disrupts gene expression. Techniques: RNA/DNA/protein extraction, SDS-PAGE, Western blotting, PCR, principles of genetic analysis, Illumina sequencing, use of genome database resources Goal #2: Express a recombinant protein in E. coli for production of antiserum - choose a conserved region of the assigned protein for expression - clone the DNA segment encoding this region into an E. coli expression vector - express the protein and purify it by nickel affinity chromatography Techniques: Multiple sequence alignments, molecular cloning by Gibson Assembly, E. coli transformation, expression and purification of his-tagged proteins from E. coli 2

3 Tentative Class Schedule The schedule below shows the order of topics and assignments, but the dates may change as the term progresses due to the inevitable uncertainties of doing research. On some days we will not need the full scheduled time, but on others it may be necessary to stay late or come in outside of class hrs. Date Lecture Lab Assignments (Readings available on Canvas) Week 1 Sept 25 Course overview: biological context and goals of experiments. Lab basics: Safety, Lab Notebooks, Pipetting, Making Solutions Pipetting 101. Making solutions Introduction to your mutants (mark mutants for harvest at next class). Dorrell and Howe (2012) What makes a chloroplast? Reconstructing the establishment of photosynthetic symbioses. J. Cell Sci. 125: Sept 27 Principles of Electrophoresis & Western blotting Protein extraction and quantification. Make detailed plan for protein gels Ten things every molecular biologist should know. pp 2-11 Harvest tissue for future protein, RNA, DNA extraction. Sept 29 Review of photosynthesis. Quantify photosynthetic enzymes by SDS- PAGE/Western blotting: run gels and make blots Nelson & Ben-Shem. (2004) The complex architecture of oxygenic photosynthesis. Nat Rev Molec Cell Bio 5, Finish harvesting tissue Week 2 Oct 2 Where your mutants came from: The "PML" mutant collection Probe Western blots with first set of antibodies. Method reinforcement: Prep and quantify replicate protein samples. Belcher et al (2015) Largescale genetic analysis of chloroplast biogenesis in maize. Biochim Biophys Acta 1847: Oct 4 Run gels of new protein samples and blot Work on lab notebooks Oct 6 Molecular cloning of gene segments for protein expression in E. coli. Reprobe first Western blots with 2nd antibody cocktail Probe new western blots with first set of antibodies. Ungraded lab notebook check. Chapter Recomb DNA Technology and Molec Cloning Optional: Chapter 9.2 Molecular Cell Biology 5th edition 3

4 Week 3 Lecture Lab Assignments Oct 9 Sequence alignments; design inserts for expression constructs. order gene blocks encoding antigens. Probe new western blots Oct 11 with 2 nd set of antibodies New SDS-PAGE/blots to fill in missing protein data. Probe western #2 with final antibody Prep for day s group meeting (informal presentations using Power Point). Oct 13 Working with Protein, DNA, and RNA Group meeting: Present mutant phenotypes, western data and antigen designs LAB NOTEBOOK CHECK #1. Week 4 Oct 16 Oct 18 Chloroplast gene expression Maize genome, Transposon tagging, Mu transposons Illumina Sequencing Technology Cosegregation analysis/pedigrees Linearize plasmid for antigen expression clones Extract DNA from wt cousins for cosegregation tests Quantify extracted DNAs by nanodrop and on gels. Check plasmid digests on same gels. Discuss pedigrees QUIZ #1 Barkan (2011) Expression of Plastid Genes: Organelle-specific elaborations on a prokaryotic scaffold. Plant Phys 2011 Lisch (2015) Mutator and MULE Transposons. Microbiology Spectrum 3:MDNA g-by-synthesis-explaining-the-illuminasequencing-technology/ Oct 20 Mu-Illumina Method for Mapping Mu insertions PCR overview Organize presentations: 1. Plastids and Human Disease-the Apicoplast 2. Chloroplast-Based Biotechnology 3. Improving Photosynthesis to Feed the World Prep DNA from mutant samples. Williams-Carrier et al, (2010) Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize. Plant J 63:

5 Week 5 Lecture Lab Assignments Oct 23 Review: Gibson Assembly, bacterial transformation Practice PCRs and test quality of leaf DNA preps by determining whether they are PCRable. Quantify mutant DNA preps on gel. Oct 25 Predicting protein function: conserved domains, orthologous groups, etc. Gibson Assembly of Inserts/Vectors for protein expression, and E. coli transformation on Earnshaw (2013) Deducing Protein Function by Forensic Integrative Cell Biology, PLOS Biology e Working with Mu- Illumina insertion database. Strategies for prioritizing insertions for followup. Oct 27 Primer on Giving Scientific Presentations Pick colonies from transformation plates to a gridded plate, grow O/N and store at 4. Prep for lab notebook check. Analyze Mu-Illumina data: identify insertions that cosegregate with phenotypes (day 1) Week 6 Oct 30 PCR primer design View Illumina reads with the IGV; identify Mu insertion sites. Identify cosegregating insertions and prioritize candidates for PCR followup, day 2. LAB NOTEBOOK CHECK #2. QUIZ #2 (Gibson assembly, molecular cloning, Mu- Illumina, cosegregation analysis) Nov 1 Protein expression project: Inoculate cultures for plasmid minipreps Prepare slides for day s group meeting Design/order PCR primers to genotype top candidate insertion 5

6 Nov 3 Group Meeting: Priorities for validation by PCR (optional if time: Complications in cosegregation analysis: epigenetic suppression of phenotypes Protein expression project: Plasmid minipreps Present data and explain reasoning for choosing candidates to test by PCR. Week 7 Lecture Lab Assignments Induction/purification of his-tagged proteins Nov 6 Protein expression project: Test minipreps for presence of inserts by PCR. QUIZ #3: Take Home and Turn in on Thursday Tuesday Nov 8 OUTSIDE OF CLASS TIME Journal Club: Plastids and Human Disease: The Apicoplast (2 students) Protein expression project: Innoculate overnight cultures for induction/purification of the recombinant protein 2.5 hr before class: Use overnight cultures to inoculate new cultures and grow for 2.5 h At start of class: add IPTG to induce and grow for 3 hr Quiz #3 Due Optional background: Chakraborty (2016) Understanding the biology of the Plasmodium falciparum apicoplast. Life Sci 158:104 Nov 10 Journal club: Chloroplast-based biotechnology 3 students Pellet cells and freeze pellets Plan tests of gene-specific primers (for Tuesday) McFadden and Yeh (2017) The apicoplast: now you see it, now you don t. Intnl J for Parasitology. Amberg-Johnson et al (2017) Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens. elife 6:e29865 Purify his-tagged proteins Lab Notebook Check #3 Maliga and Bock (2011) Plastid Biotechnology: Food, Fuel, and Medicine for the 21st Century. Plant Physiol 155:1501- Fuentes et al (2016) A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. Elife 5:e13664 Zhang et al (2015) Full crop protection from an insect pest by expression of long doublestranded RNAs in plastids. Science

7 Week 8 Lecture Lab Assignments SDS-PAGE of purified recombinant Nov 13 proteins Nov 15 PCR tests: Gene Specific Primers PCR validations: Genotype mutants and WT cousins with the winning primers, day 1 Nov 17 Journal Club: Improving Photosynthesis to Feed the World Extract RNA from mutant and wildtype seedlings. PCR validations: day 2 Quantify RNA by nanodrop and agarose gel electrophoresis Ort et al (2015) Redesigning photosynthesis to sustainably meet global food and bioenergy demand. PNAS 112: Order primers of top candidate genes for RT-PCR analysis. Muller et al (2011) Nonphotochemical quenching. A response to excess light energy. Plant Phys 125: Kromdijk et al (2016) Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science 354:857- Week 9 Nov 20 PCR validations: day 3 QUIZ #4: Take Home and Turn in on MONDAY (evaluating cosegregation by PCR genotyping; recomb protein expression and purification) Nov 22 Nov 24 THANKSGIVING BREAK Week 10 Nov 27 Nov 29 day Dec 1 Dec 6 RT-PCR to assay expression of the disrupted gene suspected to underlie the mutant phenotype PCR and RT-PCR continued. Prepare for final symposium Bringing it all together: Symposium: Presentation of Results (30 min/group) Read papers relevant to your gene identifications and (for graduate students) research proposal 3 hour session, TIME TBA based on student schedules. Final Paper Due at noon. PDF or deliver hard copy to Alice s office. Seek/read literature relevant to your gene identifications and (for graduate students) research proposal 7