Holston, A. S. | Search bioRxiv

bioRxiv2026

Characterizing Sequence-Function Relationships in Chimeric DcuS/EnvZ Histidine Kinases at Scale

While bacterial sensor histidine kinases (SHKs) are widespread as natural molecular biosensors, tools for high-throughput characterization of SHK signaling phenotypes are limited, hindering wide scale implementation of bacterial-based sensing. Here, we developed a synthetic two-component signaling system that reports chimeric SHK signaling via a standardized fluorescence readout. With this synthetic system, we screened a library of chimeric DcuS/EnvZ SHKs to characterize sequence-function relationships within in the DcuS sensory and transmembrane domains. We quantified the effects of 1,173 mutations on signaling outputs in the presence of fumarate, a native DcuS ligand, as well as aspartate for which DcuS has minimal affinity for. We identified eleven positions across the DcuS domains which significantly alter aspartate responsiveness and selectivity and further observed a role for cytoplasmic N-terminal residues in determining signaling outputs. In future studies, this framework will expedite design of biosensors for novel ligands by enabling high-throughput screening of mutagenized libraries of natural SHKs.

synthetic biology↗

bioRxiv2023

Degenerate DropSynth for Simultaneous Assembly of Diverse Gene Libraries and Local Designed Mutants

Protein engineering efforts often involve the creation of hybrid or chimeric proteins, where functionality critically hinges on the precise design of linkers and fusion points. Traditional methods have been constrained by a focus on single genes or the random selection of fusion points. Here we introduce an approach which enables the creation of large gene libraries where each library comprises a multitude of diverse, specifically designed genes, each with a corresponding set of programmatically designed fusion points or linkers. When combined with multiplex functional assays, these libraries facilitate the derivation of generalized engineering principles applicable across whole protein families or domain types. Degenerate DropSynth is a multiplex gene synthesis technique which allows for the assembly of up to eight distinct variants for each of the 1,536 designed parent genes in a single reaction. We assemble chimeric sensor histidine kinases and demonstrate the assembly of genes up to 1 kbp in length with an 8% rate of perfect assemblies per gene. Our findings indicate that incorporating an increased number of variants in droplets containing barcoded beads does not significantly affect the rate of perfect assemblies. However, maintaining a consistent level of degeneracy across the library is important to ensure good coverage and reduce inequality. The results suggest the potential for scaling this process to assemble at least 8,000 distinct variants in a single reaction. Degenerate DropSynth enables the systematic exploration of protein families through large-scale, programmable assembly of chimeric proteins, moving beyond the limitations of individual protein studies.

synthetic biology↗