- Obtain E. coli cells containing the appropriate expression plasmids of wild-type enzymes.
- Prepare autoinduction media (lab 1).
- Start 40-48 hr expression of wild-type and sfGFP control protein samples.
- Determine which type of polyacrylamide gel to use with for crude protein gel (SDS-PAGE) using information about the predicted protein structure and molecular weight (lab 1).
- Prepare SDS-PAGE buffers.
- Harvest cells and prepare samples for crude protein gel (lab 2).
- Pour and run SDS-PAGE on crude samples (lab 2).
- Begin a literature search for an assay procedure to test the enzyme kinetics. Resources will be provided on Canvas.
- Analysis of protein production by SDS-PAGE
- Separate proteins on polyacrylamide gel
- Fix and stain proteins on gel
- Digitally image gel
- Read about protein purification procedures.
- Prepare purification buffers.
- Refine hypothesis and finalize selection of mutations and ncAAs for term project.
By end of Thursday of Week 2:
- Notify professor which protein, mutations, and ncAAs will be used.
Deep Thoughts on Lab Citizenship
As you embark on the experimental component on this course, it is important to consider appropriate lab citizenship. You are working in a lab space shared with many other students- not only your lab group but also the other lab groups in your class AND the lab groups in other sections, some of which are using the same bench space as you. You need to be considerate of all these people as well as any instructors and TAs when working in the lab- that’s a lot of people!
A few recommendations:
· Clean up after yourself. Clean up your bench space and any common areas of the lab immediately after you use them (this includes the balance area, refilling pipet tip boxes, etc.)
· Be courteous and respectful. Be respectful of your group members and other people in lab. Be respectful of people’s things- do not use or interfere with someone else’s experiments or reagents.
· Be prepared. You are expected to show up prepared for that day’s lab. Develop a plan ahead of time for what your group wishes to accomplish in lab and how you plan to go about accomplishing it. You have a limited amount of time in lab to work. Coming in with a plan already established is respectful to not only your other group members and the instructors but will also help you work efficiently and effectively.
· Use lab resources wisely. The reagents and supplies are expensive and your lab fees only cover a fraction of the costs of running this project-based lab.
· Label. All of your supplies should be appropriately labeled with your group names and contents. This not only assists your other group members in locating necessary reagents but also avoids the potential hazards and hassle of dealing with unknown reagents in the case of a spill or cleanup.
· Be punctual. Just as you would show up on time for a lecture course, you are expected to show up on time and prepared to work at the start of class.
· Be safe. Follow all appropriate safety procedures and ask if you have questions.