20.109(S09):Protein engineering research article

Overview

The culminating assignment for Module 1 will be a research article in which you describe your protein engineering investigation. It is essential that you relate not merely what you did but why you did it, and not only what your data presently shows but what it means for the future. The target audience for this report is a scientifically literate reader who is unfamiliar with your specific field. Thus, you can assume rapid comprehension – but not a priori knowledge – of technical information, and consequently should strive to present your work in a logical, step-by-step fashion.

For more information, see below for specific and this link for general lab report guidelines.

Collaboration and Integrity

This report must be written entirely by you. Thus, while we encourage you to discuss your results with your lab partner and other classmates, you may not share text or figures. Please do not plagiarize (accidentally or otherwise) the class wiki, or any other writing/images available online or elsewhere.

In order for you to have the best learning experience possible, and to maintain a fair playing field for all students in the class, we ask that you do not look at completed assignments for similar modules run in previous years.

Logistics

Method of Submission

Please email your completed report to 20109 DOT submit AT gmail DOT com, with filename Firstinitial_Lastname_LabSection_Mod1.doc (for example, S_Hockfield_TR_Mod1.doc).

First Draft Submission: March 10th/11th

The first draft of your research article is due by 11 am on March 10th (Tuesday) or March 11th (Wednesday), according to which day you have lab.

Revised Article Submission: April 2nd/3rd

Your first draft, with feedback from both the writing and the technical faculty, will be returned on March 19th (Thursday) or 20th (Friday). You will then have the opportunity to revise your report for up to a one and one-third letter grade improvement. In other words, a C can be revised up to an B+, a C+ to an A-, a B- to an A, etc. The final draft is due on April 2nd (Thursday) or April 3rd (Friday), according to which day you have lab.

Formatting Expectations

Your main document (excluding figures) should be/have
- .doc (preferred) or .pdf
- 12-pt font
- with 1-inch margins
- double-spaced (excepting the abstract)
Figures can be made in a separate drawing program (such as powerpoint), and should be submitted as .pdf

Guidelines on length

Not counting figures, report length should not exceed the following (concise writing is appreciated and rewarded!):

Introduction: 2-3 pages
Methods: 3 pages
Results: 3-4 pages
Discussion: 3-5 pages
Total: Your final report should be ~12-15 pages (certainly not longer than 17 pages).

Content Guidelines

Abstract

The abstract serves as a condensed version (not >250 words) of your report, from motivational background to key results (and how they were found) to implications for the future. By convention, it should be single-spaced and not include citations.

Introduction

The introduction provides a framework for the story you are about to tell (The Amazing Adventures of a Mutant Calcium Sensor), and thus serves two main purposes. For one, you must provide sufficient background information for a reader to understand the forthcoming results. Just as importantly, you must motivate the audience to keep reading! How? Reveal the significance of the work through connections to both prior scientific accomplishments and future applications. You are welcome to use your own creativity and judgement as to what a good introduction should look like; however, you may find the suggested structure and content below useful.

Paragraph 1: most general, "big picture" paragraph. Here you should introduce the reader to the broader context of your experiment and motivate why your research is important. You might address questions such as those below, but you won't necessarily touch on all of them equally or even at all. Be sure to tell a coherent story, not a dense but unconnected list of facts.
- Why is calcium biologically relevant?
- What types of natural and synthetic calcium sensors exist and why are they useful?
- What is protein engineering and by what strategies can it be accomplished?

Paragraph 2: "zooming in" somewhat. Now that the reader has a frame for thinking about your research, you can present background information in more depth, including
- The structure of inverse pericam, and particularly of calmodulin
- Specific areas of the protein that could be altered (not just the one you chose, but broad categories of modification)
- Why changing calcium (or M13) affinity or cooperativity could be useful

Paragraph 3: most specific, a description of your particular investigation. Finally you can cover topics such as
- How you chose your specific mutation (and rationale for the Y64D mutation) given the local protein structure
- Your expectations for how these mutations will affect protein function
- A brief summary of how you intend to assess whether your experiment worked
- A brief overview of your results and conclusions

Materials and Methods

The methods section should allow an independent investigator to repeat some or all of your experiments. Use sub-section headings to allow readers to quickly identify experiments of interest to them. When commercially available kits were used, it is sufficient to cite the name of the kit and say that it was used according to the manufacturer’s protocol. The key to a good methods section is developing your judgement for what information is essential and what is extraneous.

Results

The purpose of the results section is to present your data in a relatively unbiased way, but with some guiding framework. Begin with a short overview of the entire experiment, and then delve into specific sub-sections that describe each piece of the work. Note that the sub-sections should be organized by functional content, not by what you did each day in lab. One potential division might be the following: construction of the mutant plasmid, verification of mutant DNA and protein production, and characterization of the mutant protein. However, other schemes could work as well or better.

Each sub-section should begin with an overview sentence that introduces the present experiment and end with a sentence stating the primary conclusion reached from that experiment. The overview and/or concluding sentences should also provide a transition to the previous/next piece of data. You may present your reader with the broad strokes of what your data indicate, particularly in the sub-section headings and concluding sentences, and in the figure caption titles. However, you should reserve detailed interpretation of your data for the discussion section.

For the most part, this linked paper on "cameleon" calcium sensors demonstrates the approximate division of labour we expect between results and discussion. (Note that the conjecture about microdomains on p. 10557 might be better suited for discussion than results per se.) You may also find some of the topics discussed relevant for your own report.

Discussion

The purpose of the discussion section is to interpret and contextualize your data. You should begin by reiterating your major findings. Then you might do any or all of the following: connect your findings to other research (published or that of your peers); describe any ambiguities and sources of error in the data, and suggest future experiments to resolve uncertainties; explain where you expect your work may lead, and suggest specific experiments for extending your findings; describe any conceptual or technical limitations of the research. Finally, you should explain the significance of your findings to basic science and to engineering applications. Like the previous sections, the discussion should have a clear organization and narrative flow.

Suggested Figures

Your report is expected to contain more or less the following figures (not in this order). Of course you are welcome to make modifications and additions as you see fit. Figures should generally be described in the Results section.

Schematics/diagrams
- Depiction of your design strategy for mutagenesis
- Plasmid map for wild-type and mutant DNA, revealing expected sizes for diagnostic digests
Figures
- Gel with un-/digested mutant DNA, if it shows anything
- Gel with diagnostic digests
- Sequence analysis
- SDS-PAGE
- Titration curves for WT and mutant protein
Tables or just text
- Mutagenesis colony counts
- Colony counts for transformed DE3 and controls
- Cell pellet observations – colour and relative growth
- Purified protein concentrations
- Table of K_D and/or Hill values for competing models

References

You are not expected to do a thorough survey of the relevant primary literature. However, your introduction (and potentially discussion) should contain a total of at least three references. Formatting guidelines are described here.

Recommended Order of Assembly

Your data (results and figures) is the heart of your paper. Begin by assembling and understanding it as clearly as possible. Then you can write the framing material – introduction and discussion, while compiling a few relevant references. (Actually, you will write a draft of the introduction about halfway through the module, once you have begun to develop some understanding of protein engineering. The final revision is best written once you have all your results.) You should generally write the abstract last, once you have a big picture understanding of every element of your investigation. As for the methods section, you might write it in short pieces day-by-day, while the procedures are fresh in your mind.

Evaluation

The full descriptive rubric for lab reports can be found on the guidelines page. The weighting for Module 1 is shown below:

Section	Worth (%)
Title	2
Abstract	5
Introduction	20
Materials and Methods	10
Results	25
Discussion	25
References	3
Writing	10