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 procedure that are important in ensuring the reader understands your experimental approach.
   • 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.

Module 2: System engineering

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).