Engineering an <italic style="font-style: italic">in</italic> <italic style="font-style: italic">vivo</italic> directed evolution system for developing genetic switches

ZHOU Yujie; YI Xiao

doi:10.12211/2096-8280.2025-023

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Engineering an in vivo directed evolution system for developing genetic switches

Research Article | 更新时间：2026-01-15

- Engineering an in vivo directed evolution system for developing genetic switches
- Synthetic Biology Journal Vol. 6, Issue 6, Pages: 1421-1434(2025)
- 作者机构：
  
  1.中国科学院深圳先进技术研究院，定量合成生物学全国重点实验室，深圳合成生物学创新研究院，广东深圳 518055
  2.中国科学院大学，北京 100049
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2025-023
  CLC： Q819
- Received：24 March 2025，
  
  Revised：2025-04-23，
  
  Published：31 December 2025
- 稿件说明：
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周玉洁，易啸. 遗传开关的活细胞定向进化平台的建立及应用［J］. 合成生物学， 2025， 6（6）： 1421-1434

ZHOU Yujie， YI Xiao. Engineering an in vivo directed evolution system for developing genetic switches［J］. Synthetic Biology Journal， 2025， 6（6）： 1421-1434
周玉洁，易啸. 遗传开关的活细胞定向进化平台的建立及应用［J］. 合成生物学， 2025， 6（6）： 1421-1434 DOI： 10.12211/2096-8280.2025-023.

ZHOU Yujie， YI Xiao. Engineering an in vivo directed evolution system for developing genetic switches［J］. Synthetic Biology Journal， 2025， 6（6）： 1421-1434 DOI： 10.12211/2096-8280.2025-023.

摘要

在合成生物学中，转录因子作为遗传开关，可通过模块化组合策略构建复杂的调控网络。现有工程化的基因元件种类有限，因此建立高效的遗传开关定向进化平台对于基因线路设计和代谢通路优化具有重要价值。本研究整合TADR活细胞定向进化系统和基于半乳糖磷酸激酶GalK及绿色荧光蛋白GFP的双重正负筛选系统，构建了优化遗传开关的实验平台。通过转录因子TetR的进化实验验证了该平台的有效性，并成功将转录因子AcuR从阻遏模式反转为激活模式（AcuR-OFF）。AcuR-OFF蛋白的开关性能与野生型类似。相较于常用的易错PCR技术，TADR构建突变文库成本更为低廉，突变规模多样性更高。双重正负筛选系统极大降低了假阳性率，在目标种群丰度低的极端情况下仍然具有高效筛选能力。该实验平台有望成为遗传开关开发与优化的有力工具。

Abstract

Transcription factors

as genetic switches

are used in synthetic biology to construct complex regulatory networks through modular combinations. Transcription factors are classified by two groups

repressors and activators. Repressors bind DNA and repress the expression level of downstream genes without inducers but will dissociate from DNA in the presence of inducers. The regulation type of repressor is called ON system

whereas activators bind DNA only after induced. The regulation type of activator is called OFF system. The goals to optimize transcription factors include identifying new substrates

enhancing sensitivity

adjusting dynamic range

and modifying regulation types. However

current types of engineered genetic elements are limited. Consequently

establishing an efficient platform of directed evolution for developing genetic switches is important for the design of genetic circuits and the optimization of metabolic pathways. In this study

we integrated

vivo

directed evolution system of TADR (Targeted Artificial DNA Replisome) with a dual positive-negative selection system based on galactose kinase (GalK) and green fluorescent protein (GFP) to develop an experimental platform for optimizing genetic switches. The effectiveness of this platform was validated through experiments with transcription factor TetR. We successfully converted TetR from a repressor to an activator. In addition

we also converted transcription factor AcuR from a repressor to an activator (AcuR-OFF) which has not been reported before. The response of AcuR-OFF mutants to

inducer is opposite to that of the wild type

but with similar dynamic range. Compared with the commonly used error-prone PCR technique

TADR is more affordable for construction of mutants library with higher genetic diversity. The dual positive-negative selection system greatly reduces the rate of false positives

and is able to screen in extreme cases where abundance of the target mutants within the population is low. This experimental platform is expected to be a powerful tool for the development and optimization of genetic switches

thereby advancing the research in synthetic biology.

关键词

Keywords

references

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⁰

Engineering an in vivo directed evolution system for developing genetic switches

DOI：10.12211/2096-8280.2025-023

摘要

Abstract

关键词

Keywords

references