Experiment Overview
| Repository ID: | FR-FCM-Z25G | Experiment name: | qY2H 2019/06/11 | MIFlowCyt score: | 65.75% |
| Primary researcher: | David CLUET | PI/manager: | David CLUET | Uploaded by: | David CLUET |
| Experiment dates: | 2019-06-03 - 2019-06-11 | Dataset uploaded: | Jul 2019 | Last updated: | Feb 2020 |
| Keywords: | [quantitative liquid Yeast two-hybrid] [affinity ladder] [Protein Protein Interaction] [Fluorescent fusion proteins] | Manuscripts: | |||
| Organizations: |
CNRS and Ecole Normale Superieure de Lyon, Lyon, (France)
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| Purpose: | The objective is to perform a quantitative yeast 2-Hybrid analysis with 11 protein couples (6 organisms, 10 protein families). The tested affinities span between 117pM to 17µM. Bait proteins are fused with Tag RFP and LexA DNA Binding Domain (BD). Prey proteins are fused with yEGFP and B42 Activation Domain (AD). The reporter gene under the control of the LexA operator is a tandem of Tag BFP (pSB_BFP2 plasmid). An affinity ladder with dual gating on Tag RFP-H and yEGFP-H is performed. | ||||
| Conclusion: | All cumulative Tag BFP curves are ordered in agreement with the affinity of the various couples, except for BD-TEM / AD-BLIP1 that generates lower phenotype (see also Figure 5A of the manuscript and discussion section). | ||||
| Comments: | Samples of 1 million cells were generated to generate an affinity ladder with a dual gating approach: 1. Gating: Tag RFP-H: 700-900 (linear scale) 2. Gating: yEGFP-H: 5000-6000 (linear scale) | ||||
| Funding: | CNRS ENS fond Recherche | ||||
| Quality control: | Non fluorescent empty Bait and Prey couple (sample BD-NF-AD-NF.fcs) is used for the setup of the acquisition parameters. Background signal of the system is determined with the fluorescent empty Bait and Prey couple (sample BD-Empty-AD-Empty.fcs), which serves as negative control for interaction. The BD-B112 is a functional "covalent" (infinite affinity, sample BD-B112-AD-Empty.fcs) transcription factor. It is used as positive control for interaction. | ||||
Experiment variables
| Conditions | |
|---|---|
| · 100ml SGR-UHW with 0.25% Galactose and 1% Raffinose at 30°C | BD-B112-AD-Empty.fcs · BD-Barstar_D35A-AD-Barnase_H102A.fcs · BD-Barstar_D39A-AD-Barnase_H102A.fcs · BD-Barstar_W38F-AD-Barnase_H102A.fcs · BD-Barstar_WT-AD-Barnase_H102A.fcs · BD-Barstar_Y29A-AD-Barnase_H102A.fcs · BD-Barstar_Y29F-AD-Barnase_H102A.fcs · BD-Empty-AD-Empty.fcs · BD-GRB2_SH3-AD-VAV1_SH3.fcs · BD-HRas_G12V-AD-CRaf_RBD.fcs · BD-HRas_G12V-AD-CRaf_RBD_A85K.fcs · BD-NF-AD-NF .0001.fcs · BD-Nef_LAI-AD-SRC_SH3.fcs · BD-TEM-AD-BLIP1.fcs |
| Sample Type | |
|---|---|
| · EGY42a x TB50alpha diploids | BD-B112-AD-Empty.fcs · BD-Barstar_D35A-AD-Barnase_H102A.fcs · BD-Barstar_D39A-AD-Barnase_H102A.fcs · BD-Barstar_W38F-AD-Barnase_H102A.fcs · BD-Barstar_WT-AD-Barnase_H102A.fcs · BD-Barstar_Y29A-AD-Barnase_H102A.fcs · BD-Barstar_Y29F-AD-Barnase_H102A.fcs · BD-Empty-AD-Empty.fcs · BD-GRB2_SH3-AD-VAV1_SH3.fcs · BD-HRas_G12V-AD-CRaf_RBD.fcs · BD-HRas_G12V-AD-CRaf_RBD_A85K.fcs · BD-NF-AD-NF .0001.fcs · BD-Nef_LAI-AD-SRC_SH3.fcs · BD-TEM-AD-BLIP1.fcs |
| Timepoints | |
|---|---|
| · 2h | BD-B112-AD-Empty.fcs · BD-Barstar_D35A-AD-Barnase_H102A.fcs · BD-Barstar_D39A-AD-Barnase_H102A.fcs · BD-Barstar_W38F-AD-Barnase_H102A.fcs · BD-Barstar_WT-AD-Barnase_H102A.fcs · BD-Barstar_Y29A-AD-Barnase_H102A.fcs · BD-Barstar_Y29F-AD-Barnase_H102A.fcs · BD-Empty-AD-Empty.fcs · BD-GRB2_SH3-AD-VAV1_SH3.fcs · BD-HRas_G12V-AD-CRaf_RBD.fcs · BD-HRas_G12V-AD-CRaf_RBD_A85K.fcs · BD-NF-AD-NF .0001.fcs · BD-Nef_LAI-AD-SRC_SH3.fcs · BD-TEM-AD-BLIP1.fcs |
