immunoYeast & The Antibody Discovery Toolkit Specific - PowerPoint PPT Presentation

immunoYeast & The Antibody Discovery Toolkit

Specific Antibodies on Demand…So What? Now, you can... • Visualize protein localization and identify different cell types: • Precipitate proteins to find interaction partners: • Measure specific protein levels: And so much more! immunoYeast & The Antibody Discovery Toolkit

Current State of Technology immunoYeast Technology From Discovery to Production immunoYeast & The Antibody Discovery Toolkit

Make your own antibodies with immunoYeast! Obtain diversified antibody library 1. immunoYeast & The Antibody Discovery Toolkit

Error-Prone PCR Antibody Antibody Antibody Antibody immunoYeast & The Antibody Discovery Toolkit

Make your own antibodies with immunoYeast! Obtain diversified antibody library 1. Select for antibody that binds your POI 2. immunoYeast & The Antibody Discovery Toolkit

Error-Prone PCR Antibody Antibody Antibody Antigen Antigen Antigen immunoYeast & The Antibody Discovery Toolkit

� immunoYeast & The Antibody Discovery Toolkit

immunoYeast & The Antibody Discovery Toolkit

� immunoYeast & The Antibody Discovery Toolkit

Make your own antibodies with immunoYeast! Obtain diversified antibody library 1. Select for antibody that binds your POI 2. Secrete that antibody quickly and easily 3. immunoYeast & The Antibody Discovery Toolkit

Gal4 ¡ Gal80 ¡ immunoYeast & The Antibody Discovery Toolkit

immunoYeast & The Antibody Discovery Toolkit

immunoYeast The Antibody Discovery Toolkit

immunoYeast DNA Construction Progress immunoYeast & The Antibody Discovery Toolkit

How did we make our constructs? Overlap Assembly Biobrick 3A Assembly immunoYeast & The Antibody Discovery Toolkit

Overlap Assembly Need to PCR overlapping constructs You can use our overlap oligo script! immunoYeast & The Antibody Discovery Toolkit

Overlap Oligo Designer Available through NYU’s iGEM Wiki immunoYeast & The Antibody Discovery Toolkit

Overlap Oligo Designer immunoYeast & The Antibody Discovery Toolkit

to iGEM is to Learn As well as becoming cloning pros.. We learned how to Program! Processing Perl immunoYeast & The Antibody Discovery Toolkit

to iGEM is to Learn and manipulate 3D Structures! On the desktop… immunoYeast & The Antibody Discovery Toolkit

to iGEM is to Learn …And in the Weill Cornell interactive CAVE! immunoYeast & The Antibody Discovery Toolkit

Biobricked Technology Current State of Technology High-throughput in vivo Antibody Discovery Biobricks in Registry HA Tag Antibody C-Myc Tag Fragment Biobricks We Made Aga2 Aga2:linker Courtesy of iGEM Osaka Tanaka, Rabbitts, Lobato K.D. Wittrup, MIT Leeds University, UK Adimab immunoYeast & The Antibody Discovery Toolkit

Biobricked Technology High-throughput in vivo Antibody Discovery Biobricks in Registry VP16 No Interaction DNA binding domains LexBD Transcriptional Activators LexO Reporter Gene Biobricks We Made VP16 Interaction Ura3 Selective Reporter LexBD LexO Reporter Gene immunoYeast & The Antibody Discovery Toolkit

Biobricks We Biobricked Ab Discovery Toolkit Other Parts: Ura3 Selective Marker Suite of yeast promoters w/ RBS Aga2 surface display protein Flexible (Gly3)Ser4 linker Aga2 : linker 36 bp loxP site Secretion Tag immunoYeast & The Antibody Discovery Toolkit

We couldn’t have done it without: Gibson, D., Young L., Chuang, R., Venter, J.C., Hutchison, C., Smith, H. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6: 343-345 Chao, G., Lau, W.L., Hackel, B.J., Sazinsky, S.L., Lippow, S.M., Wittrup, K.D. Isolating and Engineering human antibodies using yeast surface display. Nature Protocols 1:755 – 768, 2006. Tanaka, T; Rabbitts T H. Protocol for the selection of single-domain antibody fragments by third generation intracellular antibody capture. Nature Protocols 2: 67-92, 2010. Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 7: 2087-96, 1987. immunoYeast & The Antibody Discovery Toolkit

Weill Cornell Medical School Dr. Chris Mason Yossi Steinberger NYU Dr. David Gresham Russell Durrett John Phair Harris Kaplan Sung Won Lim

Thank You! immunoYeast & The Antibody Discovery Toolkit