Week 1:
Lecture: Class logistics, overview of ClpX, MITx enrollment, electronic notebook exercise, and complete MITx Start of Semester units.
Lab: Computer lab, described in active learning module publication:
Chu, E.W.K. “Database Query to Prove Your Intuitions on Gene Predictions and Genetic Mutations.” Advances in Biology Laboratory Education, 2025, Volume 45.
Week 2:
Lecture: Faculty discussion
Lab Day 0: Lab walk-through, basic lab skills (pipetting, sterile technique, gels)
Week 3:
Lecture: Faculty discussion
Lab Day 1: Design primers for RTH mutagenesis of ClpX plasmid, prepare sterile media
Week 4:
Lecture: 7.002 & Enzyme Engineering
Lab Day 2: Set up round-the-horn PCR with mutagenic primers, prepare buffers and agarose gel
Week 5:
Lecture: Faculty discussion
Lab Day 3: Run PCRs on gel to verify amplification, purify PCR products, set up DpnI/PNK/Ligase reactions with PCR products, pouring LB+Amp Agar Plates
Week 6:
Lecture: Faculty discussion
Lab Day 4: Transform DpnI/Ligase reactions into DH5α bacteria
Week 7:
Lecture: Faculty discussion
Lab Day 5: Miniprep mutated ClpX plasmids from DH5α bacteria, send ClpX plasmid minipreps out for sequencing
Week 8:
Data Analysis Lab: Lab Day 6 Part 3.1 sequencing analysis
Lab Day 6: Analyze mutant ClpX plasmid sequence, transform mutated ClpX plasmid into expression strain, prepare purification buffer
Lab Day 6.5: Express mutant ClpX protein, collect cell pellet
Week 9:
Lecture: Faculty discussion
Lab Day 7: Analyze E. coli LOBSTR BL21(DE3) transformations, purify mutant ClpX protein, determine mutant ClpX protein concentration
Week 10:
Lecture: Faculty discussion
Lab Day 8: Run purification samples on two SDS-polyacrylamide gels, transfer one gel to PVDF membrane (western blot), incubate one gel in Coomassie stain
Week 11:
Lecture: Faculty discussion
Lab Day 9: Rehydrate, block, probe, and develop western blot, destain Coomassie-stained gel
Week 12:
Lecture: Faculty discussion
Lab Day 10: In vitro ATPase assay to test mutant ClpX activity, lab cleanup
Week 13:
Lecture: Group discussion
Lab: Student presentations: report group analysis and discuss class results
Week 14:
Class wrap up, final paper due by the following day
