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STAR Protocols · 2023 · IF 0

An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments

Xiaoqian Zeng,Shuliu Wang,Mindong Liang,Weishan Wang,Yue Jiang,Fei Xu,Leshi Liu,Hao Yan,Yaojun Tong,Lixin Zhang,Gao-Yi Tan

Summary Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al. 1

研究领域: 分子生物学 · 生物技术 · 基因工程

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ENVIRONMENT INTERNATIONAL · 2019 · IF 7.943

Genomic characterization, kinetics, and pathways of sulfamethazine biodegradation by Paenarthrobacter sp. A01

Lijia Cao,Jiayu Zhang,Renxin Zhao,Yu Deng,Jie Liu,Wenjie Fu,Yusha Lei,Tong Zhang,Xiaoyan Li,Bing Li

Biodegradation is an important route for the removal of sulfamethazine (SMZ), one of the most commonly used sulfonamide antibiotics, in the environment. However, little information is known about the kinetics, products, and pathways of SMZ biodegradation owing to the complexity of its enzyme-based biotransformation processes. In this study, the SMZ-degrading strain A01 belonging to the genus Paenarthrobacter was isolated from SMZ-enriched activated sludge reactors. The bacterial cells were rod-shaped with transient branches 2.50–4.00 μm in length with most forming in a V-shaped arrangement. The genome size of Paenarthrobacter sp. A01 had a total length of 4,885,005 bp with a GC content of 63.5%, and it contained 104 contigs and 55 RNAs. The effects of pH, temperature, initial substrate concentration and additional carbon source on the biodegradation of SMZ were investigated. The results indicated that pH 6.0–7.8, 25 °C and the addition of 0.2 g/L sodium acetate favored the biodegradation, whereas a high concentration of SMZ, 500 mg/L, had an inhibitory effect. The biodegradation kinetics with SMZ as the sole carbon source or 0.2 g/L sodium acetate as the co-substrate fit the modified Gompertz model well with a correlation coefficient (R 2 ) of 0.99. Three biodegradation pathways were proposed involving nine biodegradation products, among which C 6 H 9 N 3 O 2 S and C 12 H 12 N 2 were two novel biodegradation products that have not been reported previously. Approximately 90.7% of SMZ was transformed to 2-amino-4, 6-dimethylpyrimidine. Furthermore, sad genes responsible for catabolizing sulfonamides were characterized in A01 with high similarities of 96.0%–100.0%. This study will fill the knowledge gap in the biodegradation of this ubiquitous micropollutant in the aquatic environment.

CAS / MDL: 767-15-7

研究领域: 环境科学 · 微生物学 · 基因组学 · 生物技术

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An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments

Genomic characterization, kinetics, and pathways of sulfamethazine biodegradation by Paenarthrobacter sp. A01