20.109(S21):Module 1

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20.109(S21): Laboratory Fundamentals of Biological Engineering

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Spring 2021 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Antibody engineering        M2: Drug discovery        M3: Protein engineering       


Module 3

Lecturer: Dr. Leslie McClain
Instructors: Dr. Noreen Lyell and Dr. Leslie McClain, and Dr. Becky Meyer
Research assistant: Sarah Cowles
TAs: Jeff Hsaio and Malek Kabani

Engineering Antibodies Using Yeast Display

An antibody is a soluble or membrane bound protein, produced by immune cells in blood. In our bodies the purpose of an antibody is to recognize foreign substances, called antigens, and mark them for removal by cells that compromise our immune system. If one has a properly functioning immune system you've all been making new and improved antibodies your entire life. Antibodies are also a critical reagent in medicine, research and diagnostics. Scientists have developed methods to induce and purify antibodies from animals, and also to engineer cells to produce antibodies.

In Mod1 we'll use a method developed in the Wittrup lab to screen a library of antibodies, specifically we will screen a library of antibody fragments, called scFVs or single-chain antibody fragments. These scFvs are expressed on the exterior of yeast cells. We will select for scFVs that have improved binding to an antigen, also called a ligand. In this module we will use lysozyme as our antigen. Lysozyme is a protein that is commercially valuable and used extensively in studies of antibody-antigen binding. The screen will involve the following major steps (1) express scFV library via yeast surface display, (2) incubate scFv yeast with lysozyme, (3) collect fraction of yeast that show strong antigen binding via Fluorescence Assisted Cell Sorting (FACS), (4) harvest plasmid DNA from yeast and determine sequence of scFv plasmid, also called clone, (5) characterize single scFv clone binding to lysozyme via flow cytometry, (5) calculate binding affinity to determine if binding is improved over current scFv clones.

This module was developed thanks to the invaluable help and support of Wittrup lab PhD student, Sarah Cowles, and the generous contribution of reagents from Prof. Wittrup.


Research goal: Identify antibody fragment (scFv) that shows improved binding to the antigen, lysozyme.

Schematic overview of the experimental approach in Module 3


Acronyms and common terms-
scFv: single-chain antibody fragments
ligand: molecule that binds to another molecule, interchangeable with the term antigen in this module
FACS: Fluorescence Assisted Cell Sorting, method to measure physical properties of cells and / or fluorescence on the cells. Cells are kept alive and sorted into distinct tubes to regrow after analysis.
Flow cytometry: method to measure physical properties of cells and / or fluorescence. Cells are moved to biowaste after analysis.

Lab links: day by day

M1D1: [[]]
M1D2: [[]]
M1D3: [[]]
M1D4: [[]]
M1D5: [[]]

Assignments

References

Notes for teaching faculty

Prep notes for M3