Difference between revisions of "20.109(S16):Homework"

Module 1: Protein engineering

Due M1D1

1. Review the lab orientation exercises to prepare for the lab practical that you and your partner will complete together.
2. 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.
3. 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.
4. Complete the student registration/questionnaire from this link. Click on the "edit" tab of this page, then copy the "source code" you see to your user page, fill out the form, and print it. Submit the printout at the start of class. You do not need to keep the information on your user page after printing it.
5. Prepare for the first day of Module 1 by reading the module overview and day one introduction.

Due M1D2

1. 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!
2. Recall the prelab discussion on cloning. As you may remember, double-digests are one method used by researchers to confirm that the correct product was cloned.
   • Use your APE plasmid map to identify two double-digest approaches that will confirm your plasmid template.
   • Calculate the fragment sizes expected for each double-digest reaction. Show your work here!

Due M1D3

1. 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.

Due M1D4

1. 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 IPC.
   • Remember that schematics are figures and should include captions.
2. 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.

Due M1D6

1. 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.
2. 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.

Due M1D7

1. 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.
2. Prepare a schematic diagram that depicts your experimental approach for Module 1. See the Module 1 overview on the M1D4 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.

Due M1D8

1. 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!
2. 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.
3. 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!
     • As long as you submit your outline by M2D1, your teaching faculty will provide comments that help you prepare your mini-presentation.

Wrapping up M1

1. The Protein engineering summary draft is due by 5 pm on Saturday, March 12th for both sections.
2. The Protein engineering mini-presentation is due by 10 pm on Tuesday, March 15th or Wednesday, March 16th according to your section.
3. The Protein engineering summary revision is due by 5 pm on Monday, March 28th for both sections.
4. 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

Due M2D1

1. Prepare for the first day of Module 2 by reading the module overview and day one introduction.

Due M2D2

• Your Mod1 Abstract & Data Summary is due on Monday, March 16 at 5pm.
• Your first blog post is due within 24 hours of submitting your Abstract & Data Summary. Look for an email from Shannon inviting you to the blog.
  • Please note: you need to accept the invitation to the blog within a few days of receipt or it will expire!

We will NOT collect your final worksheet this time; it is only for your benefit.

1. Next time you will perform an assay to measure total protein concentration in your cell lysate. The reagent that we will use absorbs light at 600 nm, and the amount of protein present in the lysate will be calculated as:
   • protein concentration (μg/mL) = 100 * A₆₀₀ * 10
     • where 10 = the dilution factor used to prepare the assay
     • and 100 = the conversion factor from absorbance A to concentration
   • To save time during the next lab, you should prepare an Excel spreadsheet that will automatically calculate the volume of your lysate needed to provide between 10-20 μg of total protein.
     • The variable input to the sheet will be your measured A₆₀₀ value.
     • Your sheet will be most versatile and easy to follow if each element (such as the dilution factor) is listed and labeled separately, including units.
     • The first output of your sheet will be lysate volume. Consider that you will probably want to convert the volume to μL right away.
     • Note: you will collect two samples on M2D2. You want to load the same mass of lysate on your gel for both samples.
   • When preparing your SDS-PAGE analysis, you may only load a total volume of 24 μL on the gel and 4 μL of that volume will be sample buffer (similar to the sample buffer you used in your DNA electrophoresis). Therefore, your spreadsheet should also calculate the amount of water required to adjust the total volume to 24 μL.
   • Try doing your calculations with an A₆₀₀ of 0.71. If you'd like to check your answer in advance, just email Shannon (skalford at mit dot edu) and she'll let you know if your spreadsheet works.

Due M2D3

• Complete the activity in Part 5 of M2D2. Choose what cut type you will make by signing up on the M2D2 Talk page.
• The following calculations are required for your M2D3 lab day. We will not grade this assignment, but we will check that you have done them before class.

1. Using the NEB website, perhaps starting with the enzyme finder or another tool, plan your digest for next time. You should aim to meet the following conditions:
   • Digesting in the most optimal buffer for the enzyme or set of enzymes, at the optimal temperature
     • Note that all NEB buffers are supplied as 10X concentrates
   • Digesting with 2.5 U of each enzyme per μg of DNA
   • Digesting 7.0 μg of DNA
     • The DNA is at 907 ng/μL
   • Preparing a 25 μL total reaction volume
   • Pipetting 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 "S" size was always bought.
     • Alternatively, you can prepare a double-size (or greater) "master mix" for your reaction with excess enzyme, buffer, and water, and then mix a fraction of said mix with the DNA.
   • In sum, you should name 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.

2. Finally, recall that your primer design memo is due by 10pm on the day of your next lab session (Thursday or Friday depending on section).