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The EBY100 is an electrophoresis buffer tank designed for agarose gel electrophoresis. It is a basic piece of laboratory equipment used for the separation and analysis of DNA, RNA, or protein samples.

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Lab products found in correlation

Alexa fluor 488 labeled anti cmyc antibody, by Cell Signaling Technology (2 mentions) Pct302 vector, by Addgene (2 mentions) Alexa fluor 647 labeled anti ha antibody, by Cell Signaling Technology (2 mentions) Cytoflex, by Beckman Coulter (2 mentions) Adenine hemisulfate, by MP Biomedicals (2 mentions) Complete supplement mixture without tryptophan, by MP Biomedicals (2 mentions) C thermocellum dsm1237, by American Type Culture Collection (2 mentions) C cellulovorans, by American Type Culture Collection (2 mentions) C cellulolyticum, by American Type Culture Collection (2 mentions) R flavefaciens, by American Type Culture Collection (2 mentions) Mach1, by Thermo Fisher Scientific (2 mentions) Ammonium sulfate, by BD (2 mentions) Recombinant eby100, by BD (2 mentions) Mrna purification kit, by Thermo Fisher Scientific (1 mentions) Electromax dh10b cells, by Thermo Fisher Scientific (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Saccharomyces cerevisiae EBY100 (3 citations) S. cerevisiae EBY100 (3 citations) Saccharomyces cerevisae strain EBY100 (2 citations)

6 protocols using eby100

1

Single-Chain Affibody Dimerization on Yeast Surface

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Single chain affibody dimers were displayed on the surface of yeast S. cerevisiae strain EBY100 (Invitrogen, cat. no. C839-00) by fusion to the C-terminus of the Aga2 protein. Affibody dimers connected with a GS-linker and 3C protease cleavage site in the middle were inserted between an N-terminal cMyc epitope and a C-terminal HA tag. N-cMyc-ZA-linker-ZB-HA-C insert was cloned into the pCT302 vector (Addgene #41845). Competent yeast cells were electroporated with affibody plasmids and recovered in YPD (Sigma, cat. no. Y1375) at 30°C for an hour. Next, recovered cells were grown in SDCAA media (pH 4.5, 20 g dextrose, 6.7 g yeast nitrogen base, 5 g bactocasamino acids, 10.4 g sodium citrate and 6.4 g citric acid monohydrate dissolved in 1 liter of deionized H2O, supplemented with 10 ml of Gibco™ Penicinillin-Stereptomycin, 10,000 U/ml) to OD600 10, and the cultures were induced at 20°C for 24 hours by diluting to OD600 1.0 in SGCAA (prepared as SDCAA, but use 20g galactose instead of dextrose) (7 (link)). The display level of proteins was confirmed by staining the cells with an Alexa Fluor 488-labeled anti-cMyc antibody (Cell Signaling Technology, cat. no. 2279S) and Alexa Fluor 647-labeled anti-HA antibody (1:50 dilution; Cell Signaling Technology, cat. no. 3444S), and fluorescence was monitored by flow cytometry (Beckman Coulter, CytoFLEX).
Yang A., Jude K.M., Lai B., Minot M., Kocyla A.M., Glassman C.R., Nishimiya D., Kim Y.S., Reddy S.T., Khan A.A, & Garcia K.C. (2023). Deploying synthetic coevolution and machine learning to engineer protein-protein interactions. Science (New York, N.Y.), 381(6656), eadh1720.
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2

Yeast and Bacterial Strains for Protein Expression

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The yeast S. cerevisiae strain EBY100 (Invitrogen, Carlsbad, CA) was used for mSEA surface display. The E. coli strains (Invitrogen) Mach1 and BL21 (DE3) were used for recombinant DNA manipulation and protein expression, respectively. All recombinant yeast and E. coli strains are summarized in Supplementary Table 1. C. thermocellum DSM1237, C. cellulovorans, C. cellulolyticum and R. flavefaciens were purchased from ATCC (Manassas, VA) and cultured anaerobically following ATCC protocols. Recombinant EBY100 cells were cultured using SC-Trp medium: 1.67 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulfate (Difco Laboratories, Detroit, MI), 20 g/L glucose, 15 g/L adenine hemisulfate, and 0.64 g/L complete supplement mixture without tryptophan (MP Biomedicals, Solon, OH). Induction of aScaf display on yeast surface was performed in YPG media (1% yeast extract, 2% peptone, 2% galactose). E. coli was cultured in Luria-Bertani (LB) medium containing 50 μg/mL kanamycin. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Smith M.R., Gao H., Prabhu P., Bugada L.F., Roth C., Mutukuri D., Yee C.M., Lee L., Ziff R.M., Lee J.K, & Wen F. (2019). Elucidating structure-performance relationships in whole-cell cooperative enzyme catalysis. Nature catalysis, 2(9), 809-819.
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3

Yeast and Bacterial Strains for Protein Expression

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The yeast S. cerevisiae strain EBY100 (Invitrogen, Carlsbad, CA) was used for mSEA surface display. The E. coli strains (Invitrogen) Mach1 and BL21 (DE3) were used for recombinant DNA manipulation and protein expression, respectively. All recombinant yeast and E. coli strains are summarized in Supplementary Table 1. C. thermocellum DSM1237, C. cellulovorans, C. cellulolyticum and R. flavefaciens were purchased from ATCC (Manassas, VA) and cultured anaerobically following ATCC protocols. Recombinant EBY100 cells were cultured using SC-Trp medium: 1.67 g/L yeast nitrogen base without amino acids, 5 g/L ammonium sulfate (Difco Laboratories, Detroit, MI), 20 g/L glucose, 15 g/L adenine hemisulfate, and 0.64 g/L complete supplement mixture without tryptophan (MP Biomedicals, Solon, OH). Induction of aScaf display on yeast surface was performed in YPG media (1% yeast extract, 2% peptone, 2% galactose). E. coli was cultured in Luria-Bertani (LB) medium containing 50 μg/mL kanamycin. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Smith M.R., Gao H., Prabhu P., Bugada L.F., Roth C., Mutukuri D., Yee C.M., Lee L., Ziff R.M., Lee J.K, & Wen F. (2019). Elucidating structure-performance relationships in whole-cell cooperative enzyme catalysis. Nature catalysis, 2(9), 809-819.
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4

Single-Chain Affibody Dimerization on Yeast Surface

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Single chain affibody dimers were displayed on the surface of yeast S. cerevisiae strain EBY100 (Invitrogen, cat. no. C839-00) by fusion to the C-terminus of the Aga2 protein. Affibody dimers connected with a GS-linker and 3C protease cleavage site in the middle were inserted between an N-terminal cMyc epitope and a C-terminal HA tag. N-cMyc-ZA-linker-ZB-HA-C insert was cloned into the pCT302 vector (Addgene #41845). Competent yeast cells were electroporated with affibody plasmids and recovered in YPD (Sigma, cat. no. Y1375) at 30°C for an hour. Next, recovered cells were grown in SDCAA media (pH 4.5, 20 g dextrose, 6.7 g yeast nitrogen base, 5 g bactocasamino acids, 10.4 g sodium citrate and 6.4 g citric acid monohydrate dissolved in 1 liter of deionized H2O, supplemented with 10 ml of Gibco™ Penicinillin-Stereptomycin, 10,000 U/ml) to OD600 10, and the cultures were induced at 20°C for 24 hours by diluting to OD600 1.0 in SGCAA (prepared as SDCAA, but use 20g galactose instead of dextrose) (7 (link)). The display level of proteins was confirmed by staining the cells with an Alexa Fluor 488-labeled anti-cMyc antibody (Cell Signaling Technology, cat. no. 2279S) and Alexa Fluor 647-labeled anti-HA antibody (1:50 dilution; Cell Signaling Technology, cat. no. 3444S), and fluorescence was monitored by flow cytometry (Beckman Coulter, CytoFLEX).
Yang A., Jude K.M., Lai B., Minot M., Kocyla A.M., Glassman C.R., Nishimiya D., Kim Y.S., Reddy S.T., Khan A.A, & Garcia K.C. (2023). Deploying synthetic coevolution and machine learning to engineer protein-protein interactions. Science (New York, N.Y.), 381(6656), eadh1720.
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5

Recombinant Yersinia Invasin Expression

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Escherichia coli XL1 blue (Stratagene) was used for plasmid amplification and the Origami strain (Novagen) for protein expression. Plasmids were constructed by in vivo homologous recombination in Saccharomyces cerevisiae strain YKM2, a GFP‐expressing version of EBY100 (Invitrogen) as described previously (Moelleken and Hegemann, 2008). Yersinia pseudotuberculosis invasin was expressed as the C‐terminally His‐tagged Inv479 variant (aa 490–969) for purification of recombinant protein or as the Inv197 variant (aa 772–969) for the yeast adhesion assay as described earlier (Moelleken and Hegemann, 2008; Moelleken et al., 2010). The binding domain of OmcB (aa 40–100) from C. pneumoniae was expressed with a C‐terminal His‐tag and fused N‐terminally to GST. GST and CPn0473 were expressed as, C‐terminally His‐tagged proteins. GST‐ and His‐tagged proteins were purified with the protocols supplied by Sigma Aldrich and Qiagen respectively, and analysed on Western blots.
Fechtner T., Galle J.N, & Hegemann J.H. (2016). The novel chlamydial adhesin CPn0473 mediates the lipid raft‐dependent uptake of Chlamydia pneumoniae. Cellular Microbiology, 18(8), 1094-1105.
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6

Directional cDNA Library Construction

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Total RNA was extracted from the liver cancer cell lines using TRIzol (Invitrogen, Carlsbad, CA, USA). mRNA was obtained using the mRNA Purification Kit (Invitrogen). A random primer with a 3' NotI sequence was used to synthesize the first strand of the directional random-primed cDNA library. The second strand was synthesized using E. coli RNase H, DNA polymerase, and E. coli DNA ligase. The cDNA product was rendered blunt using T4 DNA polymerase and ligated to an EcoRI adapter. After phosphorylation with T4 polynucleotide kinase, the cDNA was digested with NotI, allowing it to be directionally inserted into pYD1 (Invitrogen) that acts as a shuttle vector between the E. coli and the Saccharomyces cerevisiae strain EBY100 (Invitrogen). Sizeselected cDNAs were fused with the pYD1 cut by EcoRI/NotI. The products were purified and transformed into ElectroMAX DH10B cells (Invitrogen) by electroporation.
Kim J.Y., Kim H.K., Jang H.J., Kim E.K, & Kim M.K. (2015). Optimization of yeast surface-displayed cDNA library screening for low abundance targets. Journal of microbiology and biotechnology, 25(4).
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