This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.To discover protein interactome maps at genomic scales, yeast two hybrid-based screening system has been used extensively in the past decade. Despite the tremendous effort in adapting liquid handling robot and array instrumentation, the current Y2H mapping approach is still labor intensive and low throughput attributed to the agar plating-based nutrient marker selection and LacZ reporter analysis. Our recent development of a yEGFP-based yeast two hybrid system enabled the quantitative analysis of GFP fluorescence of the Y2H assay cultures by flow cytometry without plating. With the recent instrumentation advancement of the HyperCyt high-throughput flow cytometry sampling platform, a plate of 384 Y2H liquid culture samples can be quantitatively analyzed in less than 20 minutes. While such system is well-suited for analyzing binary protein-protein interaction pairs, it does not have adequate throughput to perform large cDNA library screening, e.g. sorting a large human cDNA library cells (typically ~109 cells) could take days. To address this limitation, we are establishing a novel sdY2H system that incorporates the surface display reporter as an additional selection marker allowing rapid enrichment of positive cell populations via a simple magnetic separation step. This new surface reporter together with the previously established yEGFP reporter system enable rapid cDNA library screening in liquid cultures, providing a high throughput alternative to conventional plating-based screening and analysis.
| Frumkin, Jesse P; Patra, Biranchi N; Sevold, Anthony et al. (2016) The interplay between chromosome stability and cell cycle control explored through gene-gene interaction and computational simulation. Nucleic Acids Res 44:8073-85 |
| Johnson, Leah M; Gao, Lu; Shields IV, C Wyatt et al. (2013) Elastomeric microparticles for acoustic mediated bioseparations. J Nanobiotechnology 11:22 |
| Micheva-Viteva, Sofiya N; Shou, Yulin; Nowak-Lovato, Kristy L et al. (2013) c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response. BMC Microbiol 13:249 |
| Ai, Ye; Sanders, Claire K; Marrone, Babetta L (2013) Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves. Anal Chem 85:9126-34 |
| Sanders, Claire K; Mourant, Judith R (2013) Advantages of full spectrum flow cytometry. J Biomed Opt 18:037004 |
| Cushing, Kevin W; Piyasena, Menake E; Carroll, Nick J et al. (2013) Elastomeric negative acoustic contrast particles for affinity capture assays. Anal Chem 85:2208-15 |
| Piyasena, Menake E; Austin Suthanthiraraj, Pearlson P; Applegate Jr, Robert W et al. (2012) Multinode acoustic focusing for parallel flow cytometry. Anal Chem 84:1831-9 |
| Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A et al. (2012) One-dimensional acoustic standing waves in rectangular channels for flow cytometry. Methods 57:259-71 |
| Vuyisich, Momchilo; Sanders, Claire K; Graves, Steven W (2012) Binding and cell intoxication studies of anthrax lethal toxin. Mol Biol Rep 39:5897-903 |
| Chaudhary, Anu; Ganguly, Kumkum; Cabantous, Stephanie et al. (2012) The Brucella TIR-like protein TcpB interacts with the death domain of MyD88. Biochem Biophys Res Commun 417:299-304 |
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