Applied Molecular Biology Lab

Experiment 5, Lab Day 20

Parts	Pages in Lab Manual
Part 5.2: Yeast display magnetic bead sorting	114–118

Day 20 In-Lab Questions (PDF)

Pre-Lab and Post-Lab Instructions

Day 20 Post-Lab Notebook Tips and Reminders

Follow all instructions and answer all four questions below for your Day 20 post-lab:

1. State a modification you could do to your overall magnetic bead sorting protocol that would likely help increase the sensitivity of selecting binders for your SARS-CoV-2 target protein. 
2. Having decided on the conditions for your magnetic bead sorting protocol, you wish to perform a test run to assess the effectiveness of your sorting protocol. For your test run beads, you use magnetic beads coated with the c-Myc epitope peptide. You use a test starting population containing exactly:         
   2 x 10⁴ yeast cells expressing a Myc antibody on their surface         
   1 x 10⁶ yeast cells that do not express anything on their surface         
   You perform your test run bead sorting protocol using the above reagents. Your final isolated “sub-library” after this bead sorting test run contains exactly:         
   1.2 x 10⁴ yeast cells expressing a Myc antibody on their surface         
   2.5 x 10⁵ yeast cells that do not express anything on their surface         
   Calculate the percent sensitivity and percent specificity for your overall magnetic bead sorting protocol. (Please show your work.)
3. You have successfully performed your magnetic bead sorting screen and isolated a sub-library of yeast cells expressing antibody variants that potentially bind your SARS-CoV-2 target protein. Your instructor is concerned, though, that some of the binders in your sub-library may not be specific for your actual desired target (the SARS-CoV-2 protein).         
   Design two different magnetic bead sorting experiments you could perform on your sub-library to remove any potential non-specific binders from the population. For each experiment, state what type of beads you would use and explain what fraction you would save when isolating the desired population. 
4. You have isolated the plasmids from the yeast cells in your sub-library and now wish to randomly mutagenize the antibody genes on the plasmids, with the hopes of introducing mutations that might produce a variant with increased binding affinity for the SARS-CoV-2 target protein. Design an experiment to randomly mutagenize the antibody genes on your sub-library plasmids.