Sampling the Low End of Affinity

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A bacterial omega-based two-hybrid is combined with a HIS3 selectable + GFP scalable reporter so selections (10^9 transformation efficiency) enrich functionally and sorting by GFP separates weak from strong binders. Tests with Zif268 and multiple PDZ domains (Erbin, MAGI-3) show GFP correlates with known affinity shifts and recovers known partners at low GFP (low hundreds AU) using a ~0.5 mM 3-AT threshold. Throughput is ~25 selections/week and screens include a designed 50,000 human-peptide library, which recovers low-affinity interactions.

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1. 0:00Start
2. 1:30Low Affinity Interactions are Important
3. 2:40Common platforms can miss the low affinity interactions
4. 3:52Big picture, hybrid assays
5. 5:46Omega-based B1H version 1.0. DNA-binding Specificity by Bacterial one-hybrid selection
6. 9:25Selecting DNA-binding specificity
7. 10:24Will multiple reporters produce more informed DNA-binding information?
8. 10:46Finding the low affinity interactions
9. 18:19Can we recover interactions by affinity?
10. 19:35Where do we find the known interactions?
11. 20:04Similar results found with Mag3
12. 20:30The Approach
13. 21:21Summary
14. 22:11Who did it