20.109(F22):Module 1

From Course Wiki
Jump to: navigation, search
20.109(F22): Laboratory Fundamentals of Biological Engineering

Fa22 banner image v3.png

Fall 2022 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Genomic instability        M2: Drug discovery        M3: Project design       

Module 1: Genomic instability

Cancer is a disease of the genome. Cancer is caused by accumulated mutations in genes that lead to phenotypic advantages in the progression from normal to metastatic cancer. While we know a lot about the kinds of changes that cancer cells undergo, we do not yet fully understand where mutations come from in the first place. What is clear is that changes to the DNA structure (e.g., DNA damage) can cause carcinogenic mutations. Given that DNA damage causes mutations that drive cancer progression, it is important to have effective tools for measuring DNA damage. In this module, you will learn about two different approaches for quantifying DNA damage. The first method relies on antibody recognition of changes to proteins that occur as a result of cell signaling that is triggered by DNA damage. This is an indirect measure of DNA damage. The second method relies on physical changes to the DNA structure that impact its ability to migrate when electrophoresed. This is a direct measure of DNA damage. Given the potentially deadly consequences of DNA damage, it is a good thing that our cells have robust ways to repair their DNA. Being able to measure DNA damage means that we can study DNA repair, which turns out to be a very important variable when it comes to why some people get cancer, and others do not. In addition to learning many fundamental biological and engineering concepts, you will of course learn many laboratory techniques. In particular, you will learn how to perform immunofluorescence, methods for quantitative image analysis, basic statistics and molecular pathway analysis.

An important focus of this module is public health. Of particular interest is the role that DNA damage plays in cancer susceptibility. There are several Superfund Sites in Massachusetts (these are sites that have high levels of legacy contaminants caused by prior industrial activities). These types of sites typically are contaminated with multiple DNA damaging agents, which poses the potential for enhanced health risks if certain compounds work synergistically in generating DNA lesions. While there are published reports that arsenic might affect DNA repair, the possibility that this exposures might act synergistically has not been fully explored. Here, you will play the role of a public health researcher with the goal of revealing possible combinatorial effects of a toxic metal and an oxidizing agent. Results from these studies will help guide decision making with regard to environmental remediation.

In terms of specific experiments, in this module you will measure genomic instability using two techniques: a repair foci experiment (γH2AX immunofluorescence) and a high-throughput genome damage assay (CometChip). You will use these methods to assess the effect of exposure to contaminants known to cause DNA damage. Specifically, you will assess the ability of cells to repair DNA damage caused by hydrogen peroxide (H2O2) with and without exposure to arsenite (As). H2O2 is a DNA oxidizing agent that causes DNA lesions. Oxidized bases are repaired by the base excision repair (BER) pathway. In the BER pathway, the damage is repaired by removing the damaged base, cleaving the backbone, replicating across the gap and ligating the remaining nick. There is evidence that arsenic inhibits key steps in the BER pathway, namely the ligation of the backbone nick.

Research question: Does exposure to As inhibit, or decrease, repair of H2O2-induced DNA damage, raising the possibility that combined exposure is an important risk to public health?

Fa20 M1 overview v2.png

Lab links: day by day

M1D1: Learn best practices for mammalian cell culture
M1D2: Prepare and treat cells for γH2AX experiment
M1D3: Use immunoflourescence staining to assess γH2AX experiment
M1D4: Complete data analysis for γH2AX experiment
M1D5: Treat cells for CometChip assay
M1D6: Image and analyze data for CometChip assay
M1D7: Examine experimental data using statistical methods

Major assignments

Research talk
Data summary


CometChip: A high-throughput 96-well platform for measuring DNA damage in microarrayed human cells. Journal of Visualized Experiments. (2014) 92: 1-11.

CometChip: Single-cell microarray for high-throughput detection of DNA damage. Methods in Cell Biology. (2012) 112: 247-268.

Notes for teaching faculty

Prep notes for M1