Engineering an in vivo directed evolution system for developing genetic switches

doi:10.12211/2096-8280.2025-023

Abstract

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

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

CLC Number:

Q819

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.

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.

Figures/Tables 7

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

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

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

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

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.

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

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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