Engineering topics

Kosuri, Sriram

Publications and source records attributed to Kosuri, Sriram.

3 records

DOE OSTI2023

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

Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Coiled-coils have been used as a model to study the sequence determinants of specificity. However, building well-behaved sets of orthogonal pairs of coiled-coils remains challenging due to inaccurate predictions of orthogonality and difficulties in testing at scale. To address this, we develop the next-generation bacterial two-hybrid (NGB2H) method, which allows for the rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way using next-generation sequencing readout. We design, build, and test large sets of orthogonal synthetic coiled-coils, assayed over 8,000 PPIs, and used the dataset to train a more accurate coiled-coil scoring algorithm (iCipa). After characterizing nearly 18,000 new PPIs, we identify to the best of our knowledge the largest set of orthogonal coiled-coils to date, with fifteen on-target interactions. Our approach provides a powerful tool for the design of orthogonal PPIs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Transcription activator-like effectors

Provided herein are compositions, kits and methods useful in the construction of designer transcription activator-like effector (dTALE) polypeptides.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Systematic identification of cis -regulatory variants that cause gene expression differences in a yeast cross

Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5832 natural DNA variants in the promoters of 2503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, which is consistent with the action of negative selection. Causal variants were also enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

60 APPLIED LIFE SCIENCES↗