From Course Wiki
Module 1: Protein engineering
- Review the lab orientation exercises to prepare for the lab practical that you and your partner will complete together.
- Complete the required EHS Training on-line.
- There are two web-based training modules required for 20.109. They are Chemical Hygiene Training and Managing Hazardous Waste. Chemical Hygiene includes 7 sections and 6 quizzes with an estimated completion time of 1 hour, while Managing Hazardous Waste has one quiz and should take somewhat less time to complete. Both courses can be accessed through MIT's Environmental Health and Safety page, from any computer that has your MIT certificate on it.
- If you have completed EHS training in a UROP or in another lab class, you do not need to repeat the training but you do need to print out your training record to hand in.
- From the EHS training page select the second button labeled “I have EHS training requirements for an academic subject.”
- Your summary page (“My EHS Training") should show Chemical Hygiene and Managing Hazardous Waste as requirements for 20.109. Click on the purple button “Go to Web Classes” right above the training requirements section. You may stop and start the web-based courses as many times as you need to complete them; the software keeps track of your progress in the course.
- Print the certificates of completion (or your training record) and submit at the start of class.
- Open an Evernote account at this link. We recommend downloading Evernote to your computer versus operating from the web based application (although both work). You can also download Evernote to your smart phone for ease of uploading pictures and data. You should make a notebook that will be utilized as your lab notebook. Give it a name with the following convention: 20.109(S16)_Name and share that notebook with your lab instructor(s) and TA.
- Complete the student registration/questionnaire from this link. Submit the printout at the start of class.
- Prepare for the first day of Module 1 by reading the module overview and day one introduction.
- Submit the plasmid map you generated using APE of the plasmid template you will use in the site-directed mutagenesis reaction on M1D3.
- Be sure the insert is clearly labelled with the 5' and 3' ends marked.
- Include all relevant restriction enzyme sites.
- What is the expected size of the correctly cloned product? Show your math here!
- Submit your double digest calculations.
- Include the enzymes you selected.
- Calculate the fragment sizes expected following the double-digest reaction. Show your work here!
- In the laboratory we will discuss an article from the primary literature. Specifically, we will discuss the construction and analysis of the inverse pericam (IPC) multi-component calcium sensor. To prepare for this discussion, you should closely read the paper by Nagai et al that introduced IPC. For context, you are also encouraged to read the short paper by Heim, Prasher, and Tsien, in which the very first attempt to mutagenize GFP is described, and which is a fine introduction to some of the concepts and methods used in this module. Though you should read the entire paper, be prepared to specifically discuss the section assigned to your group on the M1D3 page.
- Use your favorite drawing program to create a schematic diagram that depicts the mutagenesis strategy you used to generate your pRSET-IPC X#Z mutant.
- Include only key aspects of the mutagenesis procedure that were used to generate your IPC X#Z mutant.
- Clearly note which residue was mutated within the calmodulin subunit of IPC.
- Remember that schematics are figures and should include captions.
- You will document the work you complete for Module 1 in a Protein engineering summary. To help you pace your work, and to give you feedback throughout the process, you will draft small portions of the assignment at homework. For M1D4, you will write-up the results of your confirmation double-digest.
- Review the assignment description for the Protein engineering summary.
- Complete the first slide of your Results and Interpretations section. Per the directions for the assignment, the figure, caption, and results/interpretation bullets should all fit on a single slide.
- The gel image should be clearly labelled and have an appropriate caption with only the minimal methods details needed to interpret the figure.
- Below the gel image, describe and interpret the outcomes in concise bullets. Be sure to include details about the controls that were included.
- To prepare for M1D6, which has the potential to be long, you should fill in the table "Advanced preparation for PAGE" based on the OD values you measured.
- Post your calculated values on the M1D5 Discussion page.
- For Module 2, you will document your experiments in a written methods section that will be included in the larger System engineering research article. To help prepare you for this task, as well as give you feedback early in the process, you will draft some of the methods completed in Module 1.
- Review the guidelines for writing up your research, particularly the section on Materials and Methods.
- Write a draft of the methods completed on Days 3-5 (site-directed mutagenesis, preparation of expression system, and induction of protein expression).
- As you write your methods draft, think carefully about how the individual steps fit together and group them accordingly.
- To maximize the feedback you receive on this assignment it is important that you put effort into completing the assignment to the best of your ability. Take advantage of office hours and down-time during laboratory as you prepare your homework.
- In addition to reporting your results in the Protein engineering summary, you will need to introduce your research project. For this assignment, craft topic sentences that will be used to construct the Background and Motivation section of your summary. Please include 3-5 topic sentences that provide the information a reader will need to understand your work.
- Review the Protein engineering summary assignment description.
- The topic sentences will introduce distinct topics, much like the first sentence of a paragraph. For this assignment, the topic sentence will be followed by bulleted statements that support your topic sentence.
- In addition to the topic sentences, submit the references from which you obtained the information contained within your topic sentence.
- You should include the title of the referenced article and a brief summary of the article that includes why you chose that reference to support your topic sentence.
- Prepare a schematic diagram that depicts your experimental approach for Module 1. See the Module 1 overview page for an example, but do not simply copy this image!
- Think about which steps are important to the overall goal of your approach and which are minor methods details that can be omitted.
- Remember, schematics are figures and should have captions.
- Post your data to the M1D7 Discussion page. Include all of the requested information in the table for your group.
- All data must be posted by 1 pm to ensure everyone has access to the information and can use it for the Protein engineering summary!
- Revise your Methods section draft incorporating the feedback you received from the teaching faculty.
- In addition, include the methods completed on Day 6 (purify protein).
- As before, think critically about how to best incorporate the additional experimental methods.
- Optional assignment -- In addition to the Protein engineering summary, each student will individually complete a mini-presentation that is focused on orally presenting the data generated in Module 1. This is your opportunity to develop your oral communication skills before the Journal Club presentation in Module 2.
- Review the assignment description for the Protein engineering mini-presentation.
- For this assignment, prepare an outline for your mini-presentation that includes the following:
- Introduction to big picture that is then focused down to your research question
- Key results
- Take-home message that relates back to the big picture.
- Though this is an optional assignment, it would be best to take full advantage of it!
- As long as you submit your outline by M2D1, the teaching faculty will provide comments that help you prepare your mini-presentation.
Wrapping up M1
- The Protein engineering summary draft is due by 5 pm on Saturday, March 12th for both sections.
- The Protein engineering mini-presentation is due by 10 pm on Tuesday, March 15th or Wednesday, March 16th according to your laboratory section.
- The Protein engineering summary revision is due by 5 pm on Monday, March 28th for both sections.
- Please remember to contribute your reflections, comments, and ideas to the 20.109 class blog within 24 hr of submitting the Protein engineering summary revision.
Module 2: System engineering
- Prepare for the first day of Module 2 by reading the module overview and day one introduction.
- The oral communication assignment for this module is a Journal Club presentation that will be completed independently. To practice the skills you learned concerning slide construction during the BE Communications Lab workshop, you will craft one slide using data from the Allalunis-Turner et al. research article you reviewed in the laboratory.
- Select Fig. 3, 4, 5 OR 6 and use the information provided within the article to craft one slide that presents the data clearly and concisely to your audience.
- You may include more than one figure on your slide, but only if it is sensible to do so (i.e. if the figures support the same conclusion).
- The Journal club presentations are scheduled for M2D4 and M2D8. The slots for each day are first come, first serve; however, you should consider your workload in the coming weeks rather than simply choosing to present on M2D8 to give yourself more time...or simply choosing M2D4 to get the assignment out of the way.
- Reserve a slot to present on either M2D4 or M2D8 here.
- In addition, begin reviewing the journal article options. When you find one that you would like to present, reserve the article according to the directions here.
- Complete the exercise in Part 5 of M2D2, using this pMAX-EGFP-MCS ApE sequence.
- Submit a schematic diagram of the NHEJ reporter plasmid with all features relevant to the NHEJ assay labelled.
- As a reminder, schematic diagrams are figures and should include a caption.
- Also, choose which cut type you will assess using your NHEJ assay by signing up on the M2D2 Discussion page.
- The following calculations are required for the M2D3 exercise.
- Using the NEB website, perhaps starting with the enzyme finder or another tool, plan your digest for next time. You should meet the following conditions:
- Digest in the most optimal buffer for the enzyme(s) and at the optimal temperature.
- Note that all NEB buffers are supplied at a 10X concentration.
- Digest with 2.5 U of each enzyme per μg of DNA.
- Digest 7.0 μg of DNA.
- The DNA is at 2026 ng/μL.
- Prepare a 25 μL total reaction volume.
- Pipet no less than 1 μL of enzyme at a time.
- Note that you may need to prepare an intermediate dilution of some enzymes. To determine stock concentration, note that the laboratory stocks are the "S" options for each enzyme.
- Alternatively, you can prepare a double-size (or greater) "master mix" for your reaction with an excess of enzyme, buffer, and water, and then add an appropriate fraction of this mix to the DNA.
- In sum, you should list the reaction buffer, the reaction temperature, and the volumes of DNA, of buffer, of enzyme, and of water that you will use for your reaction.
- During Module 1, the methods section was omitted from the Protein engineering summary. Unfortunately this blissful state of affairs cannot last forever! For this assignment, you will write the first part of the methods section for your System engineering research article. This early draft will include only the procedures from Days 1-3.
- Be sure to review the information concerning the Methods section in the guidelines for writing up your research before you begin; doing so may save you some effort. Furthermore, look back at the comments you received on your Methods homework in Module 1!
- As you compose your methods for the first three days (cell culture, Western blot analysis, DNA damage), do your best to think ahead about the scope of the experiment and how D1 and D2 fit into that overall context. For example, you probably want to establish your cell strains and general culture conditions just once.
- Recall that the Module 2 assignment will be done individually and with no formal revision, so it's even more important than before that you (a) complete each homework and (b) put forth your best effort – in order to get meaningful feedback that you can use later on.
- At this point, you have a lot of practice crafting figures and captions. One change between Module 1 and Module 2 is that your associated results text will be written in paragraphs, rather than in bullet points. Another change is that you will be describing protein and cell assays, in addition to the ubiquitous DNA gel. For M2D5, prepare a figure and caption depicting your Western blot results, as well as the associated results narrative. Here are some things to keep in mind when drafting your Results section:
- Each sub-section should have a title that states the conclusion of that particular experimental step. In addition, each results paragraph should begin with an overview sentence that motivates and introduces the experiment.
- What did you do and why did you do it?
- State the results of the experiment, minimizing any interpretation of the data (save that for the Discussion!).
- Describe your figure in words.
- Be sure end each results sub-section with a concluding sentence that provides a transition to the next piece of data when possible -- stick to one topic per paragraph, but each sub-section might have a few paragraphs.
- To help prepare for M2D5, we recommend that you read the M2D5 transfection protocol and create an automated calculator in advance of class.
- Though this will not be collected, it will prove to be a helpful tool.
- For this assignment you will apply the skills you have developed in crafting methods sections to edit the methods section of a peer. Carefully read through the methods section of your classmate and comment on the following:
- Is the information complete? Are any procedural steps absent? Is important information pertaining to any of the procedures missing?
- Is the information clear? Are the procedural steps presented in an order that makes sense?
- Are the sections divided and grouped appropriately?
- Is the information presented in a way that can be followed by someone less familiar with the experimental procedure? Does the author use concentrations to convey amount?
- Are the sections written in complete sentences?
- Please be specific in your comments to the author (use examples from the text to support your comments). Feel free to clearly write directly on the printout or include your comments as a separate document, like the teaching faculty.
- The major assessment for this module will be the System engineering research article describing your project. For this assignment, you will draft the introduction of your research article. Recall that the introduction provides a framework for the story you are about to tell, with respect to both background and motivation. You are encouraged to revisit the guidelines for writing up your research before you begin.
- Consider the following questions before you start:
- Of what might the big picture section consist? Will you focus on NHEJ reporters in general, the utility of measuring NHEJ specifically, or ... what?
- What background information will the reader need to understand your project?
- What is your hypothesis?
- The first paragraph of your introduction should be written in complete sentences (don't forget your references). The remaining paragraphs can be in outline form, but must include topic sentences and a bulleted list of the supporting information that will be used to craft your paragraphs (again, don't forget your references).
- Note: Though you do not have your data yet, you should indicate where this information will be included.
- Prepare a schematic diagram that depicts your NHEJ assay.
- Include a title and a short caption for the diagram.
- Carefully read the Dietlein et. al. paper in preparation for a discussion with Prof. Samson on M2D7.
- Please review the guiding questions on the M2D7 page.
- Prepare a figure for your Module 2 System engineering research article that includes your flow cytometry plots illustrating how the various gates were established for your experiment AND one representative experimental data plot (from a repair condition).
- Your figure will have at least three sub-panels and should be prepared such that its size is no more than 1/3 of a page. Remember to label axes so that they are descriptive and make sense to the audience.
- Be sure to draft a figure caption and the associated Results section text.
Wrapping up M2
- The System engineering research article is due by 5 pm on Monday, April 18th for both sections.
- Please remember to contribute your reflections, comments, and ideas to the 20.109 class blog before 5pm on Tuesday, April 19th.
Module 3: Biomaterials engineering
- Prepare for the first day of Module 3 by reading the module overview and day one introduction.
- The primary assignment for this experimental module will be for you to develop a research proposal and present your idea to the class. For next time, please describe five recent findings that could potentially define an interesting research question. You should hand in a 3-5 sentence description of each topic, in your own words, and also formally cite an associated reference from the scientific literature. The topics you pick can be related to any aspect of the class, i.e. DNA, protein, or biomaterials engineering. During lab next time, you and your partner will review the topics and narrow your choices, identifying one or perhaps two topics for further research.
- Please note: for this assignment, you do not need to have a novel research idea completely sketched out; you simply have to describe five recent examples of existing work that you find interesting. However, you can start to brainstorm how to expand those topics into something new if you want to get ahead of the game.
- Discuss the potential research topics you prepared for the previous assignment with your co-investigator (your lab partner) and write a paragraph concerning the research question you would like to pursue for your research proposal. Please include 2-3 sentences that introduce your topic and a brief discussion of your potential plan. Consider the following as you discuss your potential research topics:
- 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.
- Can your proposal be carried out in a reasonable amount of time and with non-infinite resources?
- Take advantage of downtime in lecture and lab to discuss your research ideas with Prof. Belcher and the teaching faculty.
- Please note: The idea you submit for this assignment does not have to be the idea you present at the end of Module 3. It is okay if you change directions and decide to pursue other research questions during the process of developing your proposal.
- Consider the feedback you and your co-investigator received in the peer review exercise and begin to refine your research proposal by sharing a page (i.e. wiki, Googledoc, Evernote) to collect your ideas and resources (you can do this on one page with your partner or split the effort and each turn in an individual page).
- Keep in mind that your presentation to the class will ultimately need:
- 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
- You can organize your wiki page along these lines or however you feel is most helpful -- check out the “yeast rebuild” or the “T7.2” wiki pages on OpenWetWare for examples of research ideas in process. For this assignment, please submit a printed copy of your research page, making sure it defines your general topic (background and significance), your specific idea (research gap and general approach), and two or more references you've collected and summarized. Keep in mind that your idea may still change - if you come up with something that you like better later on, that's fine.
- For this module, you will also prepare a short Biomaterials engineering mini-report with your partner. To get you started, work with your laboratory mate to prepare an outline of the Background and Approach section for this assignment. Keep in mind your background should reference previous scientific work so there should also be a works cited or references section to this outline.
- Review the assignment details and carefully consider the questions specified within the evaluation criteria as you craft the Background and Approach section.
- Read the introductory material for M3D5, and then, based on the mass of the cathode you constructed, determine the number of mA to apply in order to fully discharge the battery in 10 hours. Use the loading factor of 17.8 mA/g, a value that is in accordance with cathodes tested during the pilot experiments for this module, and assume 63% of the mass you measured for the nanowires is active material in the cathode.
- Remember, when you generated your cathode material your composition was 70% active material (nanowires), 25% Super P, and 5% PTFE. You also need to account for the proportion of phage in your active material, which we will assume is 10%. With these values taken into account, your cathode is composed of 63% active material.
Due (after) M3D5
- The Biomaterials engineering mini-report is due by 10 pm on Thursday, May 5th or Friday, May 6th according to your laboratory section.
Wrapping up M3
- The Research proposal presentation slides are due by 1 pm on Tuesday, May 10th or Wednesday, May 11th according to your laboratory section.
- Please remember to contribute your reflections, comments, and ideas to the 20.109 class blog before 11am on Saturday, May 14th.