Fig. 1: Design and validation of the NGB2H assay. | Nature Communications

Fig. 1: Design and validation of the NGB2H assay.

From: A multiplexed bacterial two-hybrid for rapid characterization of protein–protein interactions and iterative protein design

Fig. 1

A Top: schematic of the next generation bacterial two-hybrid (NGB2H) system cloning and construct. T25, T18 - adenylate cyclase halves; BC - unique DNA barcode identifying the protein pair. Bottom: Workflow of NGB2H system. Interacting proteins reconstitute adenylate cyclase, producing cAMP (cyclic adenosine monophosphate), which drives the gene expression of the barcoded super-folder green fluorescence protein (sfGFP) reporter. Relative barcode abundance is quantified using next-generation sequencing (NGS). B The CC0 Library is composed of 16 coiled-coils tested against one another. (Bottom) Sequence logo representing the diversity in the CC0 Library. Residues that vary are shown in colour. C Interaction scores of CC0 library members are consistent between biological replicates (Pearson’s r > 0.98). D Two different codon usages have consistent interaction scores (Pearson’s r > 0.94, representative sample). E Interaction strength is similar (Pearson’s r > 0.92) regardless of which protein is attached to which half of adenylate cyclase. The blue line represents y = x. F Interaction scores of separately barcoded, cloned, and tested replicates are consistent (Pearson’s r > 0.98). G Published circular dichroism (CD) melting point (Tm) data. H Experimentally determined interaction scores. I CC0 library Raw data can be subsampled and still correlate well with the full dataset. Boxplot center lines represent the median, the hinges represent the 25th and 75th percentiles and whiskers represent the largest/smallest value within 1.5x it’s respective hinge for 50 subsamples with replacement of the full data. Source data are provided as a Source Data file.

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