Biotechnology and Bioprocess Engineering 2024; 29(1): 109-117  
Development and optimization of a modular two‑fragment LacI switch for enhanced biosensor applications
Hyewon Lee1,2 · Eugene Rha1 · Haseong Kim1,2,3
1 Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea
2 Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Korea
3 Graduate School of Engineering Biology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
Correspondence to: Haseong Kim
haseong@kribb.re.kr
Received: October 13, 2023; Revised: October 30, 2023; Accepted: October 31, 2023; Published online: February 19, 2024.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
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.
Keywords: Split LacI · Genetic switch · Cell-free protein expression · Transcription factor


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