Assignments

Overview

You will perform three series of experiments (called “modules”) over the course of the semester. The modules differ in both intellectual and experimental content, and in the ways that your learning will be assessed. Links to the experiments and assignments are provided below.

You will be working as pairs throughout the semester in lab, but you must submit individual written work (for both daily homeworks and major assignments) and give individual journal club presentations. You will close out the course by developing and presenting a novel research idea as a two-person team. Please read the 20.109 statement on collaboration and integrity for more detail about academic honesty in our class.

We appreciate that time management can be a difficult skill to develop, and that learning takes place on many time-scales. However, when assignments are turned in at wildly disparate times, it creates additional logistical burdens for the teaching faculty. Therefore, late work (both daily and culminating assignments) will be penalized 1/3 of a letter grade for each day late and will not be accepted after a week. We strongly recommend that you plan ahead and space out your work when possible.

Major Assignments

MODULES	TOPICS	ASSIGNMENTS	% OF FINAL GRADE	DESCRIPTIONS
1	RNA Engineering	Laboratory report	15	Assignment description and evaluation rubric Guidelines for writing up your research
		Computational analysis	5	Assignment description Data (RTF)
		Journal club presentation	10	Sign-up for articles Guidelines for oral presentations Evaluation rubric (PDF)
2	Protein Engineering	Research article	25	Assignment description and evaluation rubric Guidelines for writing up your research
3	Cell-Biomaterial Engineering	Research idea presentation	20	Assignment description Evaluation rubric (PDF)
3	Cell-Biomaterial Engineering	Data summary	5	Assignment description and evaluation rubric

Daily Work

In addition to the module assignments listed above, there are several other forms of required coursework.

Daily Lab Quizzes (5% of final grade)
- These are intended to refresh your memory about the experiment you are performing. They will not be hard and should take no more than 5-10 minutes at the beginning of lab.
Homework Assignments (8% of final grade)
- These will vary considerably in content and points associated with each assignment. You may be asked to perform a calculation, draw a conclusion, and/or make a figure using the data you have collected. The homeworks can be found in the “for next time” section of each lab day. You can work with your lab partner, friends and teaching assistants on these assignments but you will hand in individual assignments unless otherwise specified.
Laboratory Notebooks (5% of final grade)
- You will record your data on the white pages of a bound notebook. The yellow, duplicate pages will be collected and evaluated by the teaching assistants. Notebooks will be evaluated according to these guidelines.
Participation (2% of final grade)
- As a student in 20.109, you are expected to be an active participant in a scientific community. Your student colleagues, the teaching faculty, and especially your lab partner, are all your collaborators. They rely on you for timely posting of your data, and for your unique and thoughtful contributions during class. Your participation grade will ultimately be reviewed by all the teaching faculty, but as a starting point, you should fill out the self-assessment rubric once per module. (PDF)

Scientific data is communicated in many ways. Data can be shared informally through email with a collaborator or in lab group meetings. Data can also be formally communicated as publications in peer-reviewed journals or as hour-long seminars at international meetings. Successful scientific careers require both written and oral presentations, and scientific reputations are based on both. It is important to know that every presentation, no matter how informal, will build or hurt your reputation.

Seminars, group meetings, ten-minute talks, and journal clubs are all ways scientists share data orally. While the content, length and purpose of each talk varies, they share certain common elements, including organization, clarity, and proper attribution for the work.

The oral presentations you will give in this class will be ten-minute talks. Your talks will include an introduction to the topic, a presentation of data, a summary and a time to answer questions from your classmates. Realistically, only two or three ideas can be effectively conveyed in so short a time, and even that will require that you carefully plan what you will say and then practice saying it. You are expected to rehearse and ultimately deliver your talk with a timer running, and to adjust your presentation as needed to stay within the allotted time. Fairness to your classmates demands that you respect the ten-minute time limit.

In addition to the advice below, you should consult with our oral presentation instructor Atissa Banuazizi.

Things to Remember about Giving your Talk

A 10’ talk is not a 30’ talk given very fast
It will help if you memorize at least the first few sentences of your talk
Think of ways to transition from one slide to the next (“In the next slide I’ll show you some data that identifies the protein detected”)
Figure out how to work the lights, slide projector, curtains etc before you begin.
Keep the lights as bright as possible. If you have to turn the lights off for some image to be properly seen, then remember to turn the lights back on. People can and do fall asleep during dark seminars
Laser pointers or sticks should be used to direct attention to images on the screen. Be sure to always use a pointer with a specific purpose in mind, rather than constantly gesturing in the general vicinity of your slide; otherwise, the audience will not know what’s important. Don’t aim your laser pointer at anyone since it can damage a person’s eyes.

How to Deal with Nerves

Consider it excitement and turn it into enthusiasm
Remember that even the most experienced speakers get nervous right before a talk
Speak in a louder voice
Don’t speak in a monotone
Do practice your talk, which will help eliminate crutch words such as “so,” “um,” and “like”

Format

SECTION	MINUTES	NUMBER OF Slides	DO	DON’T
Introduction	~2	2-3	- Set the scene for the data you will present - introduce key concepts that the audience will need to follow along - At the beginning or end of the introduction, briefly state the overall scope and significance of the study - what is the central question and why is it interesting? - Try to summarize background material with a model slide	- Assume you are addressing experts - Give more information than is absolutely needed to understand the rest of your talk - Put too much information on each slide. You can bring in a few details as you speak if you are using PowerPoint animation
Data	~7	4-6	- Present the data in a logical sequence, letting each slide build upon the last - Include a title for each slide. The title should be the conclusion to be drawn - Make every element of your slide visible to the entire room. This means 20 point font or greater - Interpret each slide thoroughly and carefully - Point out strengths and weaknesses of the data along the way	- Read your talk. Similarly, don’t read lists from slides - Put too much information on each slide. Each slide should make only one point - Ever say, “I know you can’t read this, but…” Everything on each slide should be legible. - Be afraid to remind audience how the data fits into the overall question
Summary	~1	1	- Review each of your main “messages” - Say what the study contributed to the field	- Forget to acknowledge all contributors
Question & Answer	?	0	- Answer the question being asked. If you are unclear about the question, ask for clarification - Respect every question and questioner	- Take too long with one question. If the topic is involved, suggest you meet after the talk to discuss it more

Rehearse Your Talk Several Times

Find video examples of talks at MIT Video.

Instructions for Writing Specific Sections of a Scientific Paper
Recommended Order of Assembly for a Scientific Paper
Additional Scientific Writing Tips
Evaluation
- Content
- Style

Instructions for Writing Specific Sections of a Scientific Paper

A formal research article or less formal lab reports are the principal ways scientific data is conveyed to the rest of the scientific community and preserved for future examination. Each scientific journal has its own idiosyncrasies regarding particulars of the research article’s format, but the most common elements of a scientific article, in order of presentation, are:

Title
List of Authors
Abstract
Introduction
Materials and Methods
Results, including figures and tables
Discussion
References

The requirements for each section are outlined below. If you want more information, you can find parts of this text in an on-line collection of instructional materials used in the Purdue University Writing Lab. Other parts are inspired by Robert A. Day’s book, “How to Write and Publish a Scientific Paper” from Oryx Press. Still other content is based on the research and teaching experiences of 20.109 instructors Natalie Kuldell and Agi Stachowiak.

Title

The title should be short (about 10 words), interesting, and it should describe what you found. Avoid very broad titles (e.g., ‘DNA recombination’), and try to specify your own unique result within the limited space, while still maintaining readability of your title.

List of Authors

Indicate that you are the primary author of the report by underlining your name. Others who substantially contributed to your work, such as your lab partner, should also be listed.

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.

The importance of abstracts is increasing as more scientists are using computers to keep up with the literature. Since many search engines can only search for words in a paper’s title and abstract, these may be the only parts that many people read. The abstract may also be the way a journal’s editor decides whether to send your paper out for peer review or reject it as uninteresting and not generally relevant. Consequently, a well written abstract is extraordinarily important.

Introduction

The introduction provides a framework for the story you are about to tell, 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 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. Be sure to tell a coherent story, not a dense but unconnected list of facts.
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.
Paragraph 3: most specific, a description of your particular investigation.

Materials and Methods

The methods section should allow an independent investigator to repeat any of your experiments. Use sub-section headings to allow readers to quickly identify experiments of interest to them (e.g., “Protein conjugation to hydrogels” and “Cell culture and fluorescent labeling”). 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.

Note that the methods section should be written in the past tense, since your experiments are completed at the time you are writing your paper. This section should also be written in complete sentences and paragraphs, not in bullet point form.

You should cite the class wiki at the beginning of this section as follows: “Protocols were according to the 20.109 Spring 2010 lab wiki: URL accessed on January 1, 2020, unless otherwise noted.”

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 description of the goal and strategy of your overall experiment, and then delve into specific sub-sections that describe each piece of the work.

Titled sub-sections help create a high-level narrative and thus improve readability of dense papers. Effective sub-sections are organized by functional content, not by what you did each day in lab. For example, two successive titles might be “Conjugating lymphoid tissue-associated adhesion proteins to hydrogels” and “T cell migration in protein-coated hydrogels.” Less helpful titles for the same content would be “Hydrogel-protein reaction” and “Microscopy and velocity analysis.”

To write the results section, use the figures and tables as a guide. One approach you might take is to start by outlining, in point form, what you found, going slowly through each part of the figures. Then take the points and group them into paragraphs, and finally order the points within each paragraph. Present the data as fully as possible, including stuff that does not quite make sense at first glance. Ultimately, 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. (Sub-section headings and figure caption titles can also emphasize said conclusion.) The overview and/or concluding sentences should also provide a transition to the previous/next piece of data when possible. Within a sub-section, be sure to stick to one topic per paragraph; sub-sections will generally require a few paragraphs each.

Note that verbs in the results section are usually in the past tense. Only established scientific knowledge is written about in the present tense, “the world is round,” for example. You cannot presume that your own data are part of the body of established scientific knowledge, and so when you describe your own results, use the past tense, “a band of 1.3 kb was seen,” for example. There are, however, exceptions to this general rule. It is acceptable to say, “Table 3 shows the sizes of the DNA fragments in our preparation.” It is also acceptable to say, “In a 1991 paper, Ebright and coworkers used PCR to mutagenize DNA.”

Discussion

The purpose of the discussion section is to interpret and contextualize your data. You should begin by reiterating the purpose of your research and 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, whether or not you use sub-sections.

References

References are generally used in the Introduction and Discussion sections to support any claims that are not common knowledge. Include only those references that pertain to the question at hand. Journals vary considerably in their preferred format for the reference list. For this class, you should list the references alphabetically by the first author’s last name. Include all the authors, the paper’s title, the name of the journal in which it was published, its year of publication, the volume number, and page numbers. Please carefully follow the punctuation and format requirements. A typical reference should look like

Pavletich NP, Pabo CO. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 1991; 252:809-817

In the body of your report, this article would be cited as follows: “The Crystal Structure of the Zif268-DNA Complex has been Solved (Pavletich 1991).”

If two or more articles can be cited for this finding, then they are listed alphabetically, separated by a comma.

Figures and Tables

Some readers begin by scanning the figures first. The figures, with the legends, should provide a self-explanatory overview of your data. Decide what the data show, then create figures which highlight the most important points of your paper.

Tables are used to present repetitive data that is numerical. Graphs or illustrations, collectively called figures, are used to present numerical trends, raw data (like a picture of a gel), or a model that explains your work.

When you prepare your figures and tables, keep in mind that it is significantly more expensive for journals to publish figures and tables than text, so try to present the data in a way that is worthy of such added expense. The table below is an example of an ineffective table.

TEMPERATURE	REPEATS	CORTICAL CELLS	ION FLOW
24°C	5	+	+
24°C	5	-	-

The information in Table 1 could be presented in one sentence, such as: “In ten experiments carried out at 24°C, ion flow was detected only in the presence of cortical cells.” This is a clearer and more concise way to present the information. In addition, all tables and figures must have numbers, titles and legends.

Figure and Table Legends

Legends to the figures and tables explain the elements that appear in the illustration. Conclusions about the data are NOT included in the legends. As you write your first draft, you might state in a short simple sentence what the point of the figure or table is. In later drafts, make sure each element of the figure or table is explained. Your figure legends should be written in the present tense since you are explaining elements that still exist at the time that you are writing the paper.

Recommended Order of Assembly for a Scientific Paper

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.

Additional Scientific Writing Tips

In my experience, concentrating on the following topics tends to repay student effort.

Structure and Repetition

The readability of your paper can be substantially improved by its organization. Well chosen sub-section titles provide the reader with a distillation of the high-level points you want to make. Take the opportunity early and often to summarize major points - if you do this right, you will feel like you are repeating yourself too much! Each major section of the report should be more or less stand-alone, that is, understandable without having read the rest of your paper. Be sure and work from big to small to big (from broad points to details and back) throughout your paper. Finally, limit paragraphs to one topic apiece, splitting up longer linked ideas as needed.

Results vs. Discussion

One difficult task for budding scientific writers is determining what goes in a Results vs. a Discussion section. Even at the professional level, different scientific journals may have somewhat different expectations for how much interpretation is appropriate in the Results. A good rule of thumb in most contexts is to draw technical conclusions in the Results, but save scientific conclusions for the Discussion. An alternative framework that should yield similar writing choices is to focus on relatively certain conclusions in the Results, and save more speculative (but still supported!) conclusions for the Discussion.

Example of a technical conclusion: “Sequence alignment revealed that both mutants exhibited mutations in at least one of the expected sites and thus were consistent with the possible nucleotide changes that could occur with the library used.”

Example of a scientific conclusion: “A previously reported model proposes that the OmpR-P phosphatase reaction occurs by hydrolysis rather than direct phosphotransfer from H557 (Hsing 1997). Our mutant is consistent with a change to the structure of EnvZ that facilitates this hydrolysis reaction, thus increasing phosphatase activity while concurrently reducing kinase activity.”

Holistic View of Data

What should you do when two independent pieces of data suggest two very different conclusions about your overall experiment? The one thing you should not do is state that both conclusions are correct, in different parts of the paper, and hope that the reader will ignore the discrepancy! As a scientist, you must take a holistic view in your analyses. Is there any way to reconcile the two results? Is one method more trustworthy than the other or is there otherwise an indication to favour one conclusion over the other? If you have no way of determining which result is correct with your present data, can you propose future experiments to conclusively decide?

Concision

Writing concisely takes more effort than writing inefficient prose. It is also results in eminently more readable text that expresses your ideas more powerfully.

Precision and Detail

Being concise is not an invitation to being vague. Be sure to support your claims with specific examples, and to follow through on your ideas with sufficient detail.

Evaluation

The following table describes the features of reports that are excellent, good, and that need substantial improvement, respectively from left to right. Your own reports will be evaluated with these features in mind.

Content

SECTION	GOAL	EVALUATION
Title	To give content information to reader	- Engaging	- Appropriate	- Not enough content information or too much
Abstract	To concisely summarize the experimental question, general methods, major findings, and implications of the experiments in relation to what is known or expected.	- Key information is presented completely and in a clear, concise way> - All information is correct - Organization is logical - Captures any reader’s interest	-Sufficient information is presented in proper format - Would benefit from some reorganization - Understandable with some prior knowledge of experiment	- Some key information is omitted or tangential information is included - Some information is misrepresented - Some implications are omitted - Incorrect format is used
Introduction	To identify central experimental questions, and appropriate background information. To present a plausible hypothesis and a means of testing it.	- Relevant background information is presented in balanced, engaging way - Your experimental goals and predictions are clear and seem a logical extension of existing knowledge -Writing is easy to read -All background information is correctly referenced	- Relevant background information is presented but could benefit from reorganization - Your experiment is well described and a plausible hypothesis is given - With some effort, reader can connect your experiments to background information - Writing is understandable - Background information is correctly referenced	- Background information is too general, too specific, missing and/or misrepresented - Experimental question is incorrectly or not identified - No plausible hypothesis is given - Writing style is not clear, correct or concise - References are not given or properly formatted
Materials & Methods	To describe procedures correctly, clearly, and succinctly. Included a correctly formatted citation of the lab manual.	- Sufficient for another researcher to repeat your experiment - Lab manual cited	- Procedures could be pieced together with some effort - Lab manual cited	- Procedures incorrectly or unclearly described or omitted - Lab manual not cited
Results	To present your data using text AND figures/tables.	- Text tells story of your major findings in logical and engaging way - Figures and tables are formatted for maximum clarity and ease of interpretation - All figures and tables have numbers, titles and legends that are easy for the reader to follow	- Text presents data but could benefit from reorganization or editing to make story easier for reader - Text includes interpretation of results that is better suited for discussion section - Figures and tables are formatted to be clear and interpretable - All figures and tables have numbers, titles and legends	- Text omits key findings, inaccurately describes data, or includes irrelevant information - Text difficult to read due to style or mechanics of writing - Text difficult to read due to logic or organization - Figures and tables missing information, improperly formatted or poorly designed - Figures and tables have inadequate or missing titles or legends
Discussion	To evaluate meaning and importance of major findings	- Appropriate conclusions drawn from findings - Connections made between experimental findings - Connections made between findings and background information - Future directions considered - Writing is compelling	- Appropriate conclusions drawn from findings - Experimental limitations considered - Writing is clear	- Conclusions omitted, incorrectly drawn or not related to hypothesis. - Relationship between experimental findings and background information is missing or incorrectly drawn - Writing style and mechanics make argument difficult to follow
References	To give credit work on which your own is based	- Complete list of reliable sources, including peer-reviewed journal article(s) - Properly formatted in body of report and in reference section	- Adequate list or reliable sources - With minor exceptions, properly formatted in body of report and in reference section	- List is incomplete or includes sources not cited in body of report - List includes inappropriate sources - List not properly formatted - References not properly cited in body of report

Style

WRITING STYLE AND MECHANICS	EVALUATION
Voice	- Appropriate for audience - Consistent passive or active voice	- Too simple or too advanced - Irregular use of passive and active voice
Word Choice	- Concise - Says what you mean - Vocabulary (scientific and otherwise) used correctly - Avoid contractions and informal wording	- Verbose - Ambiguous or incorrect - Scientific or other vocabulary misused - Informal/colloquial tone
Fluency	- Sentences and paragraphs well structured - Clear topic for each paragraph and clear transitions between topics - Punctuation correct or only minor errors - Grammar correct or minor errors - Spelling correct	- Sentences repetitive or awkward - Ideas haphazardly arranged, logic connecting them not clear, paragraphs not focused on one topic each - Periods, commas, colons and semicolons misused. - Significant number of run-on sentences, sentence fragments, misplaced modifiers, subject/verb disagreements - Significant number of spelling errors
Scientific Format	- Past tense for describing new findings - Present tense used for accepted scientific knowledge and figure legends. - All sections included and properly formatted	- Misleading verb tenses - Some sections missing - Abstract not single spaced - Figures missing legends - References not properly formatted

WRITING STYLE AND MECHANICS

EVALUATION

Voice

- Appropriate for audience

- Consistent passive or active voice

- Too simple or too advanced

- Irregular use of passive and active voice

Word Choice

- Concise

- Says what you mean

- Vocabulary (scientific and otherwise) used correctly

- Avoid contractions and informal wording

- Verbose

- Ambiguous or incorrect

- Scientific or other vocabulary misused

- Informal/colloquial tone

Fluency

- Sentences and paragraphs well structured

- Clear topic for each paragraph and clear transitions between topics

- Punctuation correct or only minor errors

- Grammar correct or minor errors

- Spelling correct

- Sentences repetitive or awkward

- Ideas haphazardly arranged, logic connecting them not clear, paragraphs not focused on one topic each

- Periods, commas, colons and semicolons misused.

- Significant number of run-on sentences, sentence fragments, misplaced modifiers, subject/verb disagreements

- Significant number of spelling errors

Scientific Format

- Past tense for describing new findings

- Present tense used for accepted scientific knowledge and figure legends.

- All sections included and properly formatted

- Misleading verb tenses

- Some sections missing

- Abstract not single spaced

- Figures missing legends

- References not properly formatted

Assignment Overview
Logistics
Background Information
Part I: CLUSTAL Analysis
Part II: MEME Analysis
Part III: Mfold Analysis

Assignment Overview

Goals

Gain familiarity with a few common in silico methods available for:

Organizing RNA primary sequences into related families (CLUSTAL).
Identifying conserved sequence motifs present in RNA (MEME).
Predicting the secondary structures of single stranded RNA (Mfold).

It is worth noting here that while we will focus our analyses on RNA, CLUSTAL and MEME can also be successfully used for DNA and protein analysis. Mfold is additionally useful for single stranded DNA secondary structure predictions.

Learning Outcomes

By the end of this exercise, you should be able to:

Compile your data into a format readable by the web-based software being used for these analyses.
Interpret the outputs from these algorithms.
Formulate hypotheses based on your data interpretation for further testing using in silico and/or wet-lab experimental methods.
Appreciate some limitations associated with these methods.

Logistics

The computational analysis assignment is due by 11 am on Day 1 of Module 2.

Formatting expectations:

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

Background Information

Imagine that you have just completed your final round of SELEX, and now have sequence data obtained after the cloning step. Each sequence is derived from a single bacterial colony harboring a single plasmid, which encodes one aptamer sequence. In this exercise, you will be performing analyses of both the primary and predicted secondary structures of these (putative) aptamers. Overall, you will be trying to gain insights into the sequence and/or secondary structure patterns that help you:

Organize the aptamers from your SELEX experiment into sequence related classes; and
Formulate testable hypotheses aimed at connecting sequence and predicted structural analyses to function (heme binding in this case).

A broader context for appreciating the importance of the analyses you are undertaking relates to the fact that the initial SELEX library is extremely diverse (≥ 1013 unique sequences), with many of the sequences recovered at the end of your selection process being present initially only in single copy! So, what is so special about the set of sequences surviving the selection process? Do they share specific sequence motif(s) favoring high affinity binding to the target? Or, do they share structural features (dependent on or independent of) primary sequence composition that favor their interactions with the target? Alternatively, you could imagine that in the vast nucleotide sequence space you are exploring, there are many independent ways for a given sequence to interact with a target. In this case, you could recover sets of aptamers bearing no sequence or structural similarity to each other, but are all still capable of interacting with the target. The analyses you are about to perform can provide some insight into these fundamental questions, but as you will discover there are some important limitations.

For the purposes of 20.109, we do not expect you to know the details of how these algorithms work.

Part I: CLUSTAL Analysis

CLUSTAL is a program that finds strings of homology among primary sequences of nucleic acids. You can learn more by following the link to the new user FAQ on the CLUSTAL homepage.

Exercise

Go to the CLUSTAL Web site.
Copy and paste the sequences from the attached data file into the text box labeled: “Enter or paste a set of sequences in any supported format:”
- Note: Be sure that all the “>” characters are included. This is FASTA formatted data, which is a common way for inputting data into many of the bioinformatics software packages used today. You can read more about this data format at the CLUSTAL Web site.
Using the default settings, click the RUN button to begin your analysis.
You will see an alignment of the sequences you entered. Orient yourself to appreciate how your sequences are grouped relative to each other.
- To better appreciate the groupings, select the “View Guide Tree” button and then select the “Show as Phylogram Tree” option. You will need to scroll to the bottom of the page to see the results of this operation. You can always go back to your sequence alignment using the “Back to Main Result” button.

Questions

Why analyze only the sequence derived from the variable portion of your library?
Can you detect distinct groups based on this primary sequence analysis? Very briefly, how are members within a group related to each other?
Do you find any repeated sequences? Give at least two reasons why these might be present. Provide your rationale for what you believe to be the most likely reason for this finding.
Identify two very closely related, but non-identical sequences. Briefly describe the observed difference, and describe two ways in which these related aptamers could have originated.
Provide a brief summary of what overall your CLUSTAL analysis has helped you accomplish.

Associated Figure

Capture a screenshot of the phylogram tree and give it an informative title.

Part II: MEME Analysis

MEME is a program that identifies “statistically significant” contiguous sequences that are common to a group of DNA or protein sequences. You can learn at the MEME Overview page and associated links.

Exercise

In this part of your assignment, you will be analyzing your sequence data to determine if there are any motifs that are enriched in your selected library pool. Using the sequence data derived from the variable region of your putative aptamers, perform a MEME analysis.

Create a .txt file containing the variable region sequences derived from only your unique clones in FASTA format. Alternatively, you can paste these in as before.
Go to the MEME Web site. Choose the MEME option, and follow the directions for entering your sequences. You will need to enter a valid email address to receive a link to your data for easy future access.
Use the following settings for your analysis:
- Motif Frequency (how many occurrences of the motif you expect per sequence) = Zero or one
- Motif width (the number of contiguous bases you expect your recovered motif will contain): Minimum = 8 and Maximum = 15
- Number of motifs to discover: Use default value = 3

Questions

How do you decide which of the three observed motifs is most statistically significant and worth your further consideration? Explain briefly the meaning of the parameter(s) that helped you make your determination.
Why limit your analysis only to unique sequences here, whereas during you CLUSTAL analysis you considered all your sequence data?
- Hint: Perform a MEME analysis with 2 sequences (A = 6-3 & B = 8-1, for example). In one analysis, let your dataset include 6 repeats of A and 1 of B. In the next analysis, reverse the representation. Compare the motif characteristics you recover in both situations with that you recover when A and B are stoichiometrically represented in your dataset.
- Give two potential functional roles for your highest confidence MEME-detected motif during heme binding to your aptamers.

Associated Figure

When viewing the MEME output (HTML option), look to the left of each motif image to download it (e.g., as a .png file). Organize the 3 motifs found in your original analysis (not in question 2) into a figure. Give the figure an informative title and a brief caption, which should include the parameter values you used to make a determination of significance.

Part III: Mfold Analysis

Mfold is a program that predicts nucleic acid secondary structures by computing free energies for the potential structure-space of a given sequence. You can learn more on the Mfold homepage and in the cited literature at the bottom of that page.

Exercise

Here, you will predict the secondary structures for 6-5 and 8-12 using the full-length (constant + variable region) RNA sequence expected after transcription.

Follow the directions provided in your data file (read all the background and notes carefully) to come up with the required RNA sequences.
Go to the Mfold Web site.
Obtain folded structures for the 6-5 and 8-12 RNA sequences. Since you have a small number of sequences enter each individually, and perform your analyses sequentially.
- Use the default options, but please enter the following information:
  - A name for your sequence
  - A valid email address
- Before submitting the 8-12 sequence, highlight the location of the highest confidence conserved element detected during your MEME analysis in Part II.
  - This is achieved by going to the “Choose structure annotation” line, selecting the “high-light” option and entering the region you’d like to highlight. Enter this sequence range as xi-xn, where xi and xn are the first and last residue positions of the region you’d like to highlight. Residue 1 is the first G in your RNA sequence. For example, entering 20-50 will highlight residues 20-50. Multiple regions can be highlighted if you separate them by a comma (if needed).

Questions

In both the CLUSTAL and MEME analyses, you analyzed only the sequence derived from the variable region of your library. Here, you have included the constant regions in your analysis. Why is this is important? [Hint: Try your analysis using only the variable region, and compare the results with those obtained just prior].
Which folded structure is predicted to be more stable: 6-5 or 8-12? Explain your reasoning. Note that the same reasoning should help you decide which of the predicted 6-5 structures might be considered thermodynamically more favorable.
Can you identify distinguishable secondary structure feature(s) between 6-5 and 8-12 that might explain their different heme-binding properties?
Design an approach combining the in silico prediction methods being used in this exercise with laboratory experiment(s) that will permit testing whether your identified feature is relevant to heme binding.
A short sequence (27 bases) derived from 8-12 residues (33-59) containing the region corresponding to the conserved motif was chemically synthesized. It is capable of binding heme. Would you have predicted this based upon secondary structure predictions (take a look at the secondary structure of this fragment, too)? How do you rationalize these observations?

Associated Figure

Include images of all 5 structures you found in the exercise above and give them informative titles. You do not need to write figure captions. For the two full-length aptamers, you might highlight features that you discuss in question 3 on the images. Otherwise, just refer to them by base number.

Overview
Logistics
Content Guidelines
Evaluation

Overview

The culminating assignment for Module 1 will be a laboratory report in which you describe your RNA 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.

Be sure to review the 20.109 statement on collaboration and integrity.

Logistics

First Draft Submission

The first draft of your research article is due by 11 am on Day 1 of Module 2.

Revised Article Submission

Your first draft, with feedback from both the writing and the technical faculty, will be returned on Day 4 of Module 2 (9 days later). 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 one week later, on Day 6 of Module 4. Please highlight any substantial revisions to your text, for example, by using a different colored font.

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 be about 10-13 pages, and certainly not exceed 15 pages.

Though somewhat variable, typical section lengths might be:

Introduction: 2-3 pages
Methods: ~3 pages
Results: 3-4 pages
Discussion: 3-4 pages

Content Guidelines

Begin by reading the general guidelines for writing up your research, which describe the expectations for every section of the report, from Abstract to References. A few notes specific to Module 1 are as follows.

Introduction

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 (see also general guidelines) and content below useful. One approach you may choose is to emphasize method optimization to motivate your introduction, and to address the following guiding questions:

Paragraph 1
- What is SELEX?
- What are some benchmarks (in your own reasoning) that if attained, would maximize SELEX’s efficiency and accessibility.
- What are some key parameters to optimize in achieving your desired efficiency?
Paragraph 2
- Which parameter(s) have you chosen to investigate in your present study?
- What is the rationale for choosing to explore this parameter in the context of improving selection efficiency? (That is, how is this parameter linked to selection efficiency?)
Paragraph 3
- Why did you choose your specific conditions? [Consider this in the context of the parameter space covered as a lab section, in addition to what you are doing individually]
- Your expectations for how the outcome will vary as a function of your explored parameter space.
- A brief summary of how you intend to assess whether your experiment worked (yours individually, and pooled across your lab section).
- A brief overview of your results and conclusions.

Suggested Figures

Your report is expected to contain more or less the following figures. Of course you are welcome to make modifications and additions as you see fit. Recall that figures should generally be described in the Results section.

Schematics/diagrams
- Schematic showing overall experimental plan and main steps involved
Figures
- Gel from initial PCR
- Gel with RT-PCR samples
- Binding curves for your own set of data
Tables or just text
- Binding data (peak height and peak shift) for entire lab section (or entire class if available in time)

References

You are not expected to do a thorough survey of the relevant primary literature for this first report. However, your introduction (and potentially discussion) should contain a total of at least three references.

Evaluation

The full descriptive rubric for lab reports can be found on the guidelines for writing up your research. The weighting for Module 1 is as follows:

SECTION	WORTH (%)
Title	2
Abstract	5
Introduction	20
Materials and Methods	15
Results	25
Discussion	20
References	3
Writing	10

Once you have decided on a paper for your presentation, please “reserve” it by signing up on the class list. If you would like to discuss a paper not on the list below, please email it to the instructor’s email list with a brief description.

As you prepare your talk be sure to follow the class guidelines for oral presentations in this class.

Day 6 presentations will begin after lab work is finished. The entire class session on Day 8 will be devoted to presentations.

Paper Options

The list of papers below is provided as a guideline for the types of papers that might be relevant for your presentation. You are not limited to the primary research articles on this list. The list is provided simply to give you an idea of the kinds of subjects that could make suitable presentations for the class. Search PubMed yourself to find articles of interest to you.

Note: Some of these articles are open access, with full text online as linked in the following citations. Other papers, whose full text is available only to subscribers or subscribing institutions, are linked below to the abstracts.

Overview

The culminating assignment for Module 2 will be a research article in which you describe your protein engineering investigation. The term research article (as opposed to laboratory report) is meant to indicate your growing maturity as scientific writers, and our growing expectations of you. Your Module 2 paper should approach the quality of the primary scientific literature (excepting its lack of experiment repetition), especially with respect to explaining rather than merely documenting your observations.

Be sure to review the 20.109 statement on collaboration and integrity as you proceed.

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 The Journal of Cell Biology (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 Molecular Cell Biology (MCB) state:

“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 a lab report also apply to your research article, but keep in mind how the intention of the two written assignments differs.

Logistics

First Draft Submission

The first draft of your research article is due by 11 am on Day 1 of Module 3.

Revised Article Submission

Your first draft, with feedback from both the writing and the technical faculty, will be returned 9 days later on Day 4 of Module 3. You will then have the opportunity to revise your report for up to a one letter grade improvement. The final draft is due on Day 6 of Module 3. Please highlight any substantial revisions to your text, for example, by using a different colored font.

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 13 pages. The following rough division is recommended:

Introduction: 2-2.5 pages
Methods: 3-3.5 pages
Results: 2-2.5 pages
Discussion: 3-4 pages

Concise writing is appreciated and rewarded!

Content Guidelines

Begin by reading the general guidelines for writing up your research. A few notes specific to Module 2 are below:

Discussion and Citations

This section should realize all the good practices described in the Module 1 assignment, but do so at a more advanced level. You will be expected to cite the broader scientific literature more thoroughly than before, both to set up your investigative question in the introduction and to inform your analysis in the discussion. You should also propose specific future experiments and to otherwise show that you deeply understand the meaning and significance of your results; for example, if you have a hypothesis about why a mutation had the effect that it did, consider what follow-up experiments you might try. In addition to drawing conclusions from your own data, you are expected to spend some time considering your classmates’ data.

Suggested Figures

In most research endeavours, you will collect more data than you ultimately publish. In the spirit of writing a research article, in this assignment you should present only essential data. For example, if your sequencing reactions worked, there is no need to present the redundant diagnostic digest that you used to quickly check your construct. The suggested list of figures below should be suitable for most of your write-ups, but you are welcome to make changes with good reason.

Schematics/diagrams
- Depiction of your design strategy for mutagenesis
Figures
- SDS-PAGE
- Titration curves for WT and mutant protein
Tables or just text
- Sequence analysis
- Cell pellet observations – colour and relative growth
- Purified protein concentration
- Table of KD and/or Hill values for competing models

Evaluation

The full descriptive rubric for lab reports can be found on the guidelines for writing up your research. The weighting for Module 2, which is different than that for Module 1, is as follows:

SECTION	WORTH (%)
Title	2
Abstract	5
Introduction	20
Materials and methods	10
Results	23
Discussion	25
References	5
Writing	10

Data Summary

Overview

The culminating written assignment for Module 3 will be relatively informal. You should continue to strive for clear writing and thorough explanations, but you do not have to make beautiful figures or polish every last word. Keep in mind that the summaries you write today may be used by students during next spring’s offering of 20.109. Consider what information you would want to see as a student just beginning the third module, and then do your best to convey it.

At a minimum, you should introduce the experimental question you investigated (with links to publications if appropriate), describe your results (in text, tables, and perhaps 2-3 figures), then interpret your findings and discuss implications for future work. The introduction and discussion might only be about 1 long paragraph (or perhaps 2 shorter ones) each.

Although you need not include common methodological details, it is exceedingly important that you write down any such information unique to your experiment (number of cells analyzed, quantity of RNA used in RT-PCR, etc).

Collaboration and Integrity

For this assignment only, you and your partner may share figures and figure captions if you wish. However, all of the supporting text should be written individually.

Logistics

This assignment is due by 5 PM on your final wet lab day.

Evaluation

SECTION	WORTH (%)	CRITERIA
Background, Experimental Question, and Design	30	- Is the idea interesting and relevant? - Is previous foundational research sufficiently explained? - Is the experimental design likely to address the question?
Results and Interpretation of Data	50	- Is the description of results complete (including necessary methods details)? - Do the figures clearly convey the data? - Are interpretations of each piece of data reasonable?
Contextualizing Results and Suggestions for Future Work	20	- Is the import of the present experiment neither over- nor under-stated? - Are minor suggestions for future experimental changes likely to address any problems encountered? - Are broader suggestions for future work interesting and relevant?

SECTION

WORTH (%)

CRITERIA

Background, Experimental Question, and Design

- Is the idea interesting and relevant?

- Is previous foundational research sufficiently explained?

- Is the experimental design likely to address the question?

Results and Interpretation of Data

- Is the description of results complete (including necessary methods details)?

- Do the figures clearly convey the data?

- Are interpretations of each piece of data reasonable?

Contextualizing Results and Suggestions for Future Work

- Is the import of the present experiment neither over- nor under-stated?

- Are minor suggestions for future experimental changes likely to address any problems encountered?

- Are broader suggestions for future work interesting and relevant?

Guidelines for your 20.109 research proposal

Overview

Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. You can start by finding a handful of topics to share with your lab partner. Together you should discuss and evaluate the topics you’ve gathered. Consider them based on:

your interest in the topic
the availability of good background information
your likelihood of successfully advancing current understanding
the possibility of advancing foundational technologies or finding practical applications
if your proposal could be carried out in a reasonable amount of time and with non-infinite resources

It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but relatively quickly you and your partner should settle on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic.

A few ground rules that are 20.109 specific:

you should not propose any research question that has been the subject of your UROP or research experience outside of 20.109. This proposal must be original.
you should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a ten minute oral presentation.

Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you, but check out the yeast rebuild or the T7.2 wiki pages on OpenWetWare for examples of research ideas in process.

As you become more expert on your research topic, you’ll read a lot about it and you may feel

like there’s too much to read
like you have too many ideas and no way to map or prioritize them
like you don’t understand what you’re reading
all of the above.

One of the best ways to help frame the problem for yourself is to discuss it with someone new. You will have an opportunity during lab to talk with a person from another lab group. This person will offer you a fresh ear to consider your proposal. You can rework your proposal based on the conversations you’ve had.

Logistics

Prepare a 12 minute powerpoint talk that describes the research question you have identified, how you propose to study the question and what you hope to learn. A general outline your research proposal presentation is:

a brief project overview
sufficient background information for everyone to understand your proposal
a statement of the research problem and goals
project details and methods
predicted outcomes if everything goes according to plan and if nothing does
needed resources to complete the work
societal impact if all goes well

On the day you present your team should print out and bring two copies of your powerpoint slides. Black and white is fine and you can print more than one slide per page if you like. You should also write and print out your “talking points” into the comments box of each of the slides you’ll present. These are speaking notes for your presentation. They should include the words you’ll use to describe each slide and the transitions you’ve planned between them. For example, in a presentation last term one slide’s talking points were:

As you can see from this image, taken from a review on hydrocarbon metabolism in marine bacteria, the alcanivorax species is the first to grow in population after an oil spill, and its growth correlates with a decrease in aliphatic hydrcarbons.

After most alkanes have been degraded, the Cycloclaticus species blooms while aromatic hydrocarbon levels decrease
One thing to note is that as soon as they have done their job, both species return to their normal population levels.
One problem with using Alcanivorax and Cycloclasticus to clean oil spills, however is that they can only be found in specific locations

The next slide (transition statement) began: To remedy this, we decided to look into other bacteria into which we could move the hydrocarbon metabolic pathways

You will be graded on the integrated success of your presentation: concepts, slides, talking points, and presentation.

Browse Course Material

Course Info

Instructors

Departments

As Taught In

Level

Topics

Learning Resource Types

Laboratory Fundamentals in Biological Engineering

Overview

Major Assignments

Daily Work

Things to Remember about Giving your Talk

How to Deal with Nerves

Format

Rehearse Your Talk Several Times

Contents

Instructions for Writing Specific Sections of a Scientific Paper

Title

List of Authors

Abstract

Introduction

Materials and Methods

Results

Discussion

References

Figures and Tables

Figure and Table Legends

Recommended Order of Assembly for a Scientific Paper

Additional Scientific Writing Tips

Structure and Repetition

Results vs. Discussion

Holistic View of Data

Concision

Precision and Detail

Evaluation

Content

Style

Contents

Assignment Overview

Goals

Learning Outcomes

Logistics

Background Information

Part I: CLUSTAL Analysis

Exercise

Questions

Associated Figure

Part II: MEME Analysis

Exercise

Questions

Associated Figure

Part III: Mfold Analysis

Exercise

Questions

Associated Figure

Contents

Overview

Logistics

First Draft Submission

Revised Article Submission

Formatting Expectations

Guidelines on Length

Content Guidelines

Introduction

Suggested Figures

References

Evaluation

Logistics of Paper Sign-Up and Presentation

Paper Options

Methodology/Technology

Applications

Aptamers in Nature

Overview

Writing a “Research Article” versus a “Lab Report”

Logistics

First Draft Submission

Revised Article Submission

Formatting Expectations

Guidelines on Length