遗传开关的活细胞定向进化平台的建立及应用

doi:10.12211/2096-8280.2025-023

摘要/Abstract

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地降低了假阳性率，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。

关键词: 活细胞定向进化, 正负筛选, 遗传开关, 转录因子, 大肠杆菌

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 will bind DNA and repress the expression level of downstream genes without inducers. And they 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 in 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 repressor to activator. In addition, we converted transcription factor AcuR from repressor to 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 cheaper for construction of mutants library with higher genetic diversity. The dual positive-negative selection system greatly reduces 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 field of synthetic biology.

Key words: in vivo directed evolution, positive-negative selection, genetic switches, transcription factors, Escherichia coli

中图分类号:

Q819

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, 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, DOI: 10.12211/2096-8280.2025-023.

图/表 7

图1 搭建定向进化实验平台（a）TADR系统的组成元件；（b）TADR系统引入突变的过程，黑色和灰色片段分别为起始和终止序列；（c）GalK正负筛选标记的工作原理；（d）TADR-GalK-GFP实验平台的工作流程；（c）以阻遏蛋白为例，筛选系统GFP-GalK的表达受阻遏蛋白调控，通过改变诱导条件实现正向和负向筛选

Fig. 1 Construction of the directed evolution experimental platform(a) Components of the TADR system; (b) The process of introducing mutations by the TADR system, the black and gray segments represent the initiation and termination; (c) Taking the repressor protein as an example, in the screening system, the expression of GFP-GalK is regulated by the repressor protein, and positive and negative screening are achieved by changing the induction conditions; (d) The working principle of the positive and negative screening markers of GalK; (e) The working process of the TADR-GalK-GFP experimental platform

表1 实验使用的RBS序列

Table 1 The RBS sequence used in the experiment

RBS名称 RBS name	RBS序列 RBS sequence
RBS1	TCGAGGT
RBS2	TCCTGGT
RBS4	AGGAGGT
RBS5	GAGGAGG
RBS28	CTCGTGA
RBS166	TCCTGGA
RBS1036	TCCAGGA

表 2 多个启动子及RBS序列预测强度

Table 2 Prediction strength of multiple promoter and RBS sequences

启动子-RBS组合 Promoter-RBS groups	启动子预测强度 Promoter strength	RBS预测强度 RBS strength
Ptac-RBS2	16000	500
Pbla-RBS1	2000	3000
Pbla-RBS2	2000	500
Ptac-RBS1	16000	3000

图2 TetR进化实验平台搭建（a）酶标仪测定的荧光强度与吸光值计算可得群体平均荧光强度，记为M，比较诱导前后M的差值以反应阻遏蛋白的调控状态；（b）TetR对aTc诱导剂的响应曲线，诱导剂浓度分别为0、20、50、100 ng/µl，虚线表示两组对照，TetO组为PL-tetO-GFP底盘细胞（GFP为组成型表达），MG1655组不含GFP，两组分别代表了该系统中荧光表达强度的上限及下限；（c）诱导浓度为50 ng/µl时，诱导前后的群体荧光分布；（d）不同表达强度的TetR蛋白诱导前后的荧光强度变化，以及与对照组荧光强度的比较；（e）使用MG1655（含galk）与MG1655Δgalk 两种菌株验证GalK的正负筛选性能；（f）Pbla-RBS1-TetR对GalK正负筛的响应

Fig. 2 Construction of the TetR evolution experimental platform(a) The average fluorescence intensity of the population, denoted as M, can be calculated from the fluorescence intensity and absorbance value measured by the microplate reader. Compare the difference of M before and after induction to reflect the regulatory state of the repressor protein; (b) The response curve of TetR to the aTc inducer, with the inducer concentrations being 0, 20, 50, and 100 ng/µl respectively. The dashed lines represent the two control groups. The TetO group consists of PL-tetO-GFP chassis cells, in which GFP is expressed constitutively. The MG1655 group, by contrast, lacks GFP. These two groups represent the upper and lower limits of fluorescence expression intensity within this system, respectively; (c) When the induction concentration is 50 ng/µl, the population fluorescence distribution before and after induction; (d) The variations in fluorescence intensity before and after the induction of TetR proteins with different expression levels, as well as the comparison of these fluorescence intensities with those of the control groups; (e) Use two strains, MG1655 (containing galk) and MG1655Δgalk, to verify the positive and negative screening performance of GalK; (f) The response of Pbla-RBS1-TetR to the positive and negative screening of GalK

图3 筛选TetR-OFF系统（a）TetR野生型与反转表型对诱导剂的响应（b）使用GalK-GFP双正负筛的筛选过程；（c）仅使用GFP作为筛选标记的筛选过程；（d）对野生型及TetR-OFF突变子的调控性能表征，蓝色为没有诱导剂时的群体荧光分布，红色为50 ng/µl aTc 诱导后的群体荧光分布

Fig. 3 Screening of the TetR-OFF system(a) The response of the wild-type TetR and the reversed phenotype to the inducer. (b) The screening process using the GalK-GFP dual positive and negative screening. (c) The screening process using only GFP as the screening marker. (d) Characterization of the regulatory performance of the wild-type and TetR-OFF mutants. The blue represents the population fluorescence distribution without the inducer, and the red represents the population fluorescence distribution after induction with 50 ng/µl aTc.

表3 revTetR进化为TetR-ON的突变位点

Table 3 The mutation sites where revTetR evolved into TetR-ON.

突变工具 Mutation Tool	组别 Groups	位点突变 Mutation sites
	WT	N18Y	V20D	I22T	L60S	Others
易错PCR	1	18N	20V	22I	60L	R28H, T202A
易错PCR	2	18N	20V	22I
TADR	3	18N	20V	22I		Y93C, D95E
TADR	4			22I

图4 搭建实验平台开发AcuR-OFF（a）以MG1655为荧光负对照，TetO组为荧光正对照，优化AcuO调控系统；（b）分别对两组不同表达强度的TetR测定诱导浓度梯度下的荧光群体分布，以选择最佳诱导条件；（c）GalK筛选前后，没有诱导剂时目标种群比例的变化，蓝色表示GalK筛选前群体荧光分布，红色表示GalK筛选后群体荧光分布；（d）对野生型及AcuR-OFF突变子的调控性能表征，蓝色为没有诱导剂时的群体荧光分布，红色为3 mM Acr诱导后的群体荧光分布

Fig. 4 Construction of the experimental platform for the development of AcuR-OFF.(a) Using MG1655 as the fluorescence negative control and the TetO group as the fluorescence positive control, the AcuO regulatory system was optimized. (b) The fluorescence population distributions of two groups of TetR with different expression intensities were measured under the induction concentration gradients respectively to select the optimal induction conditions. (c) The changes in the proportion of the target population in the absence of the inducer before and after GalK screening. The blue color represents the population fluorescence distribution before GalK screening, and the red color represents the population fluorescence distribution after GalK screening. (d) Characterization of the regulatory performance of the wild type and the AcuR-OFF mutant. The blue color represents the population fluorescence distribution in the absence of the inducer, and the red color represents the population fluorescence distribution after induction with 3 mM Acr.

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