The research goal of our class is to determine if the S. pombe orthologs of S. cerevisiae MET genes can complement deficiencies in S. cerevisiae met strains. During the semester, you have been collecting data and organizing the data into figures and tables. You will assemble these draft versions into a final report. The data hopefully form a nice story that answers your research question and/or suggests additional experiments.
The final report should be in the form of a scientific publication, which includes the numbered sections below. Unlike the mini-reports, the Results and Discussion sections in the final report should be separate. As you write, keep in mind that good scientific writing is precise and concise! In the professional world, journals provide an economic incentive for brevity by levying page charges on authors- the longer the report, the higher the page costs to the authors! Be careful to observe the page maximum for each section.
Experiments to include in the final report include:
- Growth of your strains on selective media
- Yeast colony PCR
- Restriction map of your overexpression plasmids
- Replica plates of transformed strains
- SDS-PAGE and western blot analysis of cell extracts
- Independent experiment (placed where most appropriate within the text)
You are welcome to cut and paste from your earlier micro-reports. After all, it is your work and you are welcome to reproduce it! As you do so, be well-advised to incorporate the suggestions of your TA to improve the presentation. The only data that should be included in the final report is data that your team has generated. You should NOT include data from experiments that has been posted by other students on the data sharing site.
Include the title and abstract, as well as your name and section assignment
1. Title - Describe your project in 150 characters or less.
2. Abstract (1 paragraph, 150 words maximum) - Single-spaced
The abstract is a very brief summary of your work that should be comprehensible to the non- expert. The abstract should include the goals of your experiments, some mention (not descrip- tion) of the methods used in the experiments, a succinct summary of your experimental results, and your overall conclusions. Avoid specific details of the experiments. A reader should be able to read the abstract and understand the overall experiment and results without reading the rest of the report. Compose the abstract AFTER the rest of the report has been completed.
BODY OF THE REPORT - Double-spaced (Note that this is different from the abstract.)
3. Introduction (1.5-2 pages)
The introduction provides the context to your experiments. What is the goal of your experiments? How does your experiment fit in with what’s already known about your topic? Has anyone done experiments similar to yours before? To answer these questions, an introduction provides the reader with relevant background information derived from the scientific literature. Discuss the function of the enzyme and its evolutionary conservation. Use the format of FEMS Yeast Research to insert citations into your report. Include at least 7 citations from the scientific literature.
In the last paragraph of the introduction, give the reader a preview of your report. Tell the reader what experimental approaches you used to answer your question. End this section with one or two sentences summarizing your conclusions.
4. Materials and Methods (2-3 pages)
During the course of the semester, you have used a variety of techniques, which have been the subject of your micro-reports. In publications, the M&M section is usually divided into sub- sections, often with their own subheadings, reflecting the different experiments and techniques.
The first paragraph in an M&M section often contains details about the organisms used in the experiments and their culture conditions. When yeast and other microorganisms are used for the experiments, a genotype table is often included. Include a genotype table with table with strain and plasmid information as Table 1. Consult Cordente et al. (2009) for a good example. Include the three YMP strains that you worked with at the beginning of the semester in this table. Our YMP strains were derived from strain BY4742, which has the genotype Matα his3∆1 leu2∆0 ura3∆0. Each of the YMP strains has a met deletion caused by the insertion of a KANR gene. Themet deletion alleles should be referenced as: met#:: KANR. (See Chapter 6.)
The plasmids for this report are based on pYES2.1 and pBG1805, both of which drive expression using the GAL1 promoter. To describe these overexpression plasmids, use the format:
pBG1805-GAL1:MET# or pYES2.1-GAL1:Met#
Briefly summarize the methods used in the experiments with appropriate subheadings. Feel free to cut and paste from previous micro-reports. Add additional information for your independent experiment. Remember - if you are using a published technique (or reagent), you do not need to repeat all the details in the M&M section. Instead, you may refer to the published procedure or formulation. In this course, you can refer to a protocol in the lab manual, but you need to include any modifications that you have made to the procedures or recipes.
Rule of thumb: A good M&M section should provide enough information for a trained profes- sional to reproduce your experiments.
5. Results - (2-3 pages, not including tables and figures)
The Results section tells a story. It provides a brief narrative that guides the reader through the figures and tables with the data. Smooth transitions are important in the Results section. The reader should be able to grasp the reasoning that led from one experiment to the next. Do NOT discuss the results in any depth in this section - there is a separate discussion section in this report. Provide just enough of your conclusions that the transition to the next experiment is a logical one. For example, your selective plating data influenced your choice of primers for the yeast colony PCR experiment. (Your tables actually include some of these conclusions.) Because different kinds of experiments are frequently used in a paper, authors frequently use subheadings in the Results section.
Figures and tables from the micro-reports should be given numbers that correspond to their order of appearance in the text. PLACE the figures and table within the Results sectionNEAR where they are referenced. (Because of page breaks, it is not always possible to place the figure or table on the same page as it is referenced. Do NOT simply attach the tables and figures to the end of the report. The Cordente et al. (2009) paper provide examples of numbering and placement. Follow the general guidelines for micro-reports. Remember that a trained biologist should be able to understand your results by simply “looking at the pictures.”
6. Discussion - 2 pages maximum
Our goal this semester was to determine if Met protein functions were conserved between
S. cerevisiae and S. pombe, using complementation to analyze protein function. As you write this report, address the following issues:
Did you observe any differences in the abilities of S. cerevisiae and S. pombe genes to complement the met mutation in your strain? Is your complementation data consistent with conservation?
Reconcile the complementation data with the SDS-PAGE and western blot results. Were Met proteins of the expected size expressed in transformed cells? How does complementa- tion relate to expression of the Met protein?
If you did not observe complementation, was this because of technical issues or because of protein divergence between the two yeasts? How would you test this?
How did your independent experiment confirm or extend your results?
What additional experiments should be done to prove to the scientific community that MetXp function is/is not conserved between the two yeast species?
7. References - Single-spaced
List the references using the FEMS Yeast Research format, as you did in the bibliography assignment.