Development and optimization of a modular two-fragment LacI switch for enhanced biosensor applications

• Published in: Biotechnology and bioprocess engineering 2024, 29(1), , pp.109-117
• Main Authors: Lee, Hyewon, Rha, Eugene, Kim, Haseong
• Format: Article
• Language: English
• Subjects: Bioengineering; Biosensors; Biotechnology; Chemistry; Chemistry and Materials Science; Cloning; Drug development; Enzymes; Gene expression; Genetic engineering; Glycerol; Industrial and Production Engineering; Isopropyl thiogalactoside; Lactose; Lactose repressor; Ligands; Modular design; Optimization; Protein expression; Protein interaction; Proteins; Research Paper; Sensors; Synthetic biology; Transcription factors; 생물공학
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-024-00020-w

Being able to perform modular design of artificial transcription factors is useful in bioengineering and synthetic biology, particularly in the development of biosensors and therapeutics. This study aimed to develop a two-fragment transcription factor system by splitting a lactose repressor (LacI). To fragment LacI, we screened potential split positions from transposon-based insertional libraries that we generated to identify those positions that did not disturb the intrinsic activity of LacI. The interaction of protein tags fused with fragments induces the reassembly of LacI and recovers the isopropyl-β-D-thiogalactoside-dependent regulatory function. The split LacI-based biosensor was implemented on an in vitro platform using a cell-free protein expression system to facilitate accurate analytical studies with high reproducibility. This versatile platform holds great potential to realize the rapid and simple detection of protein–protein interactions in cell-free systems; thus, it can be further extended to disease diagnosis, particularly at the point-of-care.