Nguyen Ba Lab members publish their first first authored paper in G3

UTM Biology and The Nguyen Ba Lab are pleased to announce that Keerath Bhachu, Kortni Kindree, and Claire Chochinov (MSc graduate) have co-authored their first first-authored research paper, published in the journal G3. The authors contributed equally to the study, titled “Deep-mutational scanning libraries using tiled-region exchange mutagenesis.”

In this paper, the authors present Tiled-Region Exchange (T-REx) Mutagenesis, a novel and multiplexed adaptation of the EMPIRIC mutagenesis approach. This method simplifies the generation of deep-mutational scanning libraries by enabling parallel cloning and a one-pot Golden Gate reaction, followed by barcode fusion for downstream phenotyping. The study demonstrates that T-REx Mutagenesis is both efficient and accessible, offering a practical new tool for gene and protein function analysis.

Abstract: The analysis of gene function frequently requires the generation of mutants. Deep-mutational scanning (DMS) has emerged as a powerful tool to decipher important functional residues within genes and proteins. However, methods for performing DMS tend to be complex or laborious. Here, we introduce Tiled-Region Exchange (T-REx) Mutagenesis, which is a multiplexed modification of the EMPIRIC mutagenesis approach. Self-encoded removal fragments are cloned in parallel in non-overlapping gene locations and pooled. In a one-pot reaction, oligonucleotides are then swapped with their corresponding self-encoded removal fragments in bulk using a single Golden Gate reaction. To aid in downstream phenotyping, the library is then fused with unique DNA barcodes using the Bxb1 recombinase. We demonstrate this approach and its optimizations, to show that it is both easy to perform and efficient. This method offers simple and expedient means to create comprehensive mutagenesis libraries.

Congratulations to Keerath, Kortni, and Claire on this significant milestone in their research careers!