Difference between revisions of "20.109(S21):Module 1"

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(Engineering Antibodies Using Yeast Display)
(Engineering Antibodies Using Yeast Display)
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==Engineering Antibodies Using Yeast Display==
 
==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.
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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.  
  
Antibodies are also a critical reagent in medicine, research and diagnostics. Scientists have found ways to induce and purify antibodies from animals, and to also engineer cells to produce specific antibodies. In Mod1 we'll use a method developed in the Wittrup lab to screen a yeast library of antibodies, against a particular antigen, using Fluorescence Assisted Cell Sorting, and try to identify a single antibody that shows improved antigen binding in a quantitative binding assay.  
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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 a scFVs or a 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, (4) harvest plasmid DNA and determine sequence of scFv, (5) characterize single scFv clone binding to lysozyme via flow cytometry, (5) calculate binding affinity to determine if binding is improved.  
  
 
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.  
 
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.  
  
  
<font color = #0d368e>'''Research goal:  '''</font color>
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<font color = #0d368e>'''Research goal:  '''</font color> Identify antibody fragment (scFv) that shows improved biding to lysozyme. 
  
 
[[Image: Sp21 overview.jpg|thumb|center|700 px|Schematic overview of the experimental approach in Module 3]] <br>
 
[[Image: Sp21 overview.jpg|thumb|center|700 px|Schematic overview of the experimental approach in Module 3]] <br>

Revision as of 18:53, 25 January 2021

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 a scFVs or a 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, (4) harvest plasmid DNA and determine sequence of scFv, (5) characterize single scFv clone binding to lysozyme via flow cytometry, (5) calculate binding affinity to determine if binding is improved.

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 biding to lysozyme.

Schematic overview of the experimental approach in Module 3


Lab links: day by day

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

Assignments

References

Notes for teaching faculty

Prep notes for M3