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中国生物工程杂志

China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (4): 101-111    DOI: 10.13523/j.cb.2211055
    
Application of the Efficient Counter-selection Method in Gene Deletion of Microorganism
YANG Yi-ying1,LI Xiao-le1,LI Zi-long2,LI Qiu-yuan3,CHEN Wei1,**,YIN Shou-liang1,**
1. College of Life Sciences, North China University of Science and Technology, Tangshan 063210, China
2. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
3. Zhongrong Technology Corporation Ltd., Tangshan 064499, China
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Abstract  

With the coming of the post genome era, simple and efficient gene editing tools and methods have played a critical role in studying the regulation of gene expression and the genetic modification of engineered strains. The counter-selection methods are much convenient and efficient for gene knockout, which are based on the principle of homologous recombination. Knockout or insert of one target gene is achieved by the loss or supplement of one selective genetic marker, which generally does not rely on the traditional antibiotics. To achieve genetic manipulation of more target genes, the same selected marker can be used repeatedly over the progress of successive manipulation rounds. Because of its versatility, simplicity, efficacy, and wide range of applications, the counter-selective system has been applied in many fields of research, such as biotechnology, genetic engineering, and fundamental and applied biology. In recent years, the types, mechanisms and applications of counter-selection marker genes in microbial gene knockout experiments are reviewed. This genome-editing technique has a valuable advantage that particularly attracts the scientist’s mind, as it allows genome editing in multiple biological systems, and supports great opportunities for the future development of microbial science and rapid remodeling of genes.



Key wordsGene editing      Counter-selection methods      Gene knockout      Screening labels      Molecular genetics     
Received: 29 November 2022      Published: 04 May 2023
ZTFLH:  Q939  
Cite this article:

YANG Yi-ying, LI Xiao-le, LI Zi-long, LI Qiu-yuan, CHEN Wei, YIN Shou-liang. Application of the Efficient Counter-selection Method in Gene Deletion of Microorganism. China Biotechnology, 2023, 43(4): 101-111.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2211055     OR     https://manu60.magtech.com.cn/biotech/Y2023/V43/I4/101

Fig.1 Partial steps of the participation of PyrF in nucleotide synthesis[10]
Fig.2 Process screening target gene knockout mutants by endogenous counter-selection system based on pyrF marker gene
Fig.3 Partial steps of the participation of CodA in nucleotide synthesis
Fig.4 Process of screening target gene knockout mutants by exogenous counter-selection system based on codA marker gene
基因名称 功能 应用 参考文献
rpsL 编码链霉素的核糖体亚基蛋白(S12),与链霉素结合后使细菌蛋白的合成受到抑制 大肠杆菌、百日咳杆菌、耻垢分枝杆菌 [15]
pheS 编码苯丙氨酰-tRNA 合成酶 α 亚基,该基因缺失对苯丙氨酸及其类似物敏感 干酪乳杆菌 [32]
upp 编码UPRT,催化5-氟尿嘧啶最终转变成5-氟尿嘧啶单脱氧核糖核苷酸,抑制胸腺嘧啶的合成而导致细菌死亡 枯草芽孢杆菌 [21]
blaI blaI是β-内酰胺酶负调控基因,编码一种抑制因子抑制启动子Pbla转录表达 地衣芽孢杆菌 [22-23]
yoeBVp 转录表达大肠杆菌毒素YoeBVp,导致猪链球菌死亡 猪链球菌 [11]
thyA 编码胸腺酸合成酶,使其对甲氧苄氨嘧啶和相关类似物敏感 大肠杆菌、乳酸乳球菌、霍乱弧菌 [33]
sacB 编码蔗糖果聚糖酶,催化蔗糖水解,最终形成高分子量果聚糖,对菌体产生致死作用 大肠杆菌、嗜肺军团菌、菊欧文氏菌 [30]
lacY 编码乳糖通透酶,使细菌对邻硝基苯-β-D-半乳糖苷敏感 环状芽孢杆菌 [34]
tetAR 编码TetAR蛋白,对四环素具有耐药性,但对亲脂化合物(镰刀菌酸和喹啉酸)敏感 尖孢镰刀菌 [24]
ccdB 编码毒性蛋白质,抑制DNA促旋酶活性,导致细胞死亡 大肠杆菌、灿烂弧菌 [35?-37]
galK 编码半乳糖激酶,分解乳糖为半乳糖和葡萄糖,使菌体对2-脱氧-D-半乳糖(2-deoxygalactose,2-DOG)具有敏感性,碳源分解代谢受到抑制 耻垢分枝杆菌、结核分枝杆菌 [38?-40]
pyrF 编码乳清酸核苷-5-磷酸脱羧酶,可将5-氟乳清酸代谢为毒性代谢物5-氟尿嘧啶 龟裂链霉菌、白念珠菌 [10]
codA 编码大肠杆菌胞嘧啶脱氨酶,将5-FC转化为剧毒的5-氟尿嘧啶,使菌体致死 莱茵衣藻、酵母、青蒿 [28,41 -42]
Table 1 Types and functions of counter-selected marker genes
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