Module 2: System Engineering and Protein Foundations
Lecturer: Leona Samson
Instructors: Shannon Hughes, Aneesh Ramaswamy, and Agi Stachowiak
TA: Suhani Vora
During the first module, you spent part of your time designing and testing a diagnostic tool for detecting a microorganism. Now you will have the opportunity to apply a research tool that detects a molecular process to address an interesting biological question. Specifically, you will investigate the importance of different proteins to a type of DNA repair called non-homologous end-joining, or NHEJ. Measuring DNA repair accurately and quantitatively can pave the way for certain cancer diagnostics and therapeutics.
This time the DNA engineering has been done for you: the plasmid-based research tool to measure NHEJ already exists, along with a novel version developed just for 20.109. In this second module you will instead take a systems level view, as you explore how different topologies of DNA damage are repaired by wild-type hamster (CHO) cells, CHO cells treated with a small molecule inhibitor of an NHEJ protein, and CHO cells that are natively deficient in an NHEJ protein. Along the way, you will gain additional skills in analyzing and communicating information related not only to DNA assays (as in Module 1), but also to protein and cell-level assays. The culminating experiment will utilize flow cytometry, an amazing and infinitely useful technique that measures the fluorescence of individual cells. To evaluate class-wide trends in the flow data, you will learn and use basic statistical tools. The toolkit you develop during the first two modules should leave you well poised to tackle the third and final module.
We thank Zac Nagel from Samson lab for his many contributions to the development of this module, along with Carrie Thompson for assistance with irradiation assays. We also gratefully acknowledge Integrated DNA Technologies for speedy assistance with cloning/expression troubles that we encountered during module pilots.
Module 2 Sample Data.
The two primary techniques that you will learn to carry out and interpret in this module are flow cytometry (left) and Western blotting (right). We will use flow cytometry to quantify DNA repair events, in concert with a two color co-transfection assay. We will use Western blotting to evaluate DNA repair protein levels; the sample data shows that one of the cell lines is deficient in a particular NHEJ repair protein.
Module 2 Day 1: Introduction to cell strains and plating
Module 2 Day 2: Begin Western protein analysis
Module 2 Day 3: Choose system conditions and paper discussion
Module 2 Day 4: Complete Western and prepare damaged DNA
Note: spring break occurs between Days 4 and 5.
Module 2 Day 5: Cell preparation for DNA repair assays
Module 2 Day 6: DNA repair assays
Module 2 Day 7: Data analysis
TA notes, mod 2
NHEJ system research article