20.109(F08): Protein engineering research article

Schedule for Module 2 research article

November 4th or 5th, 2008

• First draft is due by 11:00 a.m. on the day you have lab. Please turn in your research articles electronically by emailing them to nkuldell, nlerner, myapoe AT mit DOT edu. It is important that you name your files according to this convention: Firstinitial_Lastname_LabSection_Mod2.doc, for example: S_Hockfield_TR_Mod2.doc

November 20th or 21st, 2008

• Your first draft will be returned. You will have one week from the time your report is returned to address any comments and resubmit your report if you choose. Improvements can increase your grade up to one full letter grade (e.g. a B- on the first draft could become an A-). The grades on the draft and final version are *NOT* averaged.

Writing a "research article" versus a "lab report"

A quick but unscientific survey of several journal's "instructions for authors" shows some common themes that are worth considering here. For instance, the instructions from JCB say:

"To warrant publication in the JCB, a manuscript must provide novel and significant mechanistic insight into a cellular function that will be of interest to a general readership. Manuscripts containing purely descriptive observations will not be published."

Similarly, the instructions from MCBstate:

"MCB is devoted to the advancement and dissemination of fundamental knowledge concerning the molecular biology of eukaryotic cells, of both microbial and higher organisms. In most cases, reports that emphasize methods and nucleotide sequence data alone (without experimental documentation of the functional significance of the sequence) will not be considered."

Clearly the goal of published research is not merely to catalog or describe observations but to collect the information into some coherent story that advances general understanding and provides insights that others can use. This is the critical difference between a "lab report" which primarily describes your observations and the "research article" you'll write which invites you to share the insights your data gives. Here you must frame your results to address a larger question that's of general interest to the community. Many of the format instructions that applied to the lab report also apply to your research article, but keep in mind how the intention of the two written assignments differs.

General requirements

Originality

• This report must be written by you, and thus should not be written with your lab partner, though we encourage you to discuss your results with eachother.

Format

General format requirements are:

• 12 pt font
• double spaced text except for the abstract which is single spaced
• 1” margins

Required content

Several of these elements will have been drafted as homework assignments but the instructions are repeated here.

Abstract

• Please keep the number of words under 250.
• Do not include references in the abstract.
• Try drafting this section after you’ve written the rest of the report.
• If you’re truly stuck, start by modifying one crystallizing sentence from each of the sections of your report.
• Please do not plagiarize (accidentally or other) the class wiki. This applies to your entire report.

Introduction

The homework you wrote after the first day of this new module will serve at the heart of your introduction. You should add (at least) one final paragraph to narrow the information “funnel,” ending your introduction with a clear description of the problem you’re studying and the method you are using. If you would like to preview for the reader your key results and conclusions in the last sentence of your introduction, you may.

Materials and Methods

Cite the class wiki as follows: "Protocols were according to "20.109 F'08 lab wiki: URL accessed on January 1, 2020." unless otherwise noted" then subdivide the M&M section into the following

1. Yeast strains and plasmids
   • list genotypes and plasmid names when known
2. PCR
   • include primer design info here
   • include primer sequences, for tagging and for candidate verification
   • include PCR cycling conditions
3. Yeast transformation
   • include how you selected for transformants
   • include what you did to verify that TAP tag was integrated where you thought.
4. Western Analysis
   • include how cells were grown and how they were lysed
   • include details about gel and antibodies used
   • you do not have to include information about how to blot the gel, which you can assume most folks already know
5. Yeast Microarray
   • mention kits as relevant, including deviations from the wiki/published protocol if any
   • mention how many yeast and how much RNA was used
   • describe array analytical methods in results section rather than in Materials and Methods

General note for M&M: If you used any kits for any of the manipulations, it is sufficient to cite the manufacturer’s directions, e.g. “yeast were transformed according to the Q-biogene transformation kit protocol.”

Results

You are welcome to add more figures than those listed here. These are what we consider minimally necessary.

figures

Your figures should be appended to the end of your article. If the files are large, the figures can be sent together as a file, separate from the article.

• Figure 1: Growth phenotypes. If you'd like an example of a spot test figure from a published article, consider modeling your figure and legend along the lines of figure 2 from Monehan et al "Fission yeast SWI/SNF and RSC complexes show compositional and functional differences from budding yeast"Nature Structural & Molecular Biology (2008) 15, 873 - 880 here
• Figure 2: Western blot for TAP tag. If you'd like an example of a figure that considers protein levels, consider modeling your figure and legend along the lines of figure 6 from Laprade et al. "Characterization of New Spt3 and TATA-Binding Protein Mutants of Saccharomyces cerevisiae: Spt3–TBP Allele-Specific Interactions and Bypass of Spt8" Genetics(2007)177: 2007-2017 here
  • depending on the result of your Western blot you many want to also include a panel with the agarose gel result showing the TAP tagging of your strain.
• Figure 3: Microarray analytics. This should clarify your data processing approach. It could be a flow chart or a graphic of some sort.
• Figure 4: Microarray conclusions. This should highlight the most interesting findings from your array. You'll reinforce and refer to the information in this figure when you write your results text.

text

• In paragraph form, describe each figure and the observations you made.
• Divide your results section into subsections to help the reader parse the information.
• As much as possible, reserve conclusions about your data for the discussion section. Clearly an exception to this will be which of your TAP-tagged candidates was correct, as this information is critical for the next steps in the experiments.

Discussion

You should include but are not limited to

• conclusions you can draw from your work, including any uncertainties
• other data (published or personal communications) that support or contradict your conclusions
• limitations of your work, e.g. what kinds of experiments/controls/samples would have been great to include
• next experiments you would like to try to extend your findings and strengthen your conclusions

References

• Please follow instructions that are here
• Carefully format these, including a wiki citation like: "20.109 F'08 lab wiki: URL accessed on January 1, 2020."
• Do not include encyclopedias (including Wikipedia) in your reference list since these sources are insufficiently scholarly. You should find the primary references that support those summaries.