Abstract：
:Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. Escherichia coli primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Li J,Sun J,Gao X,Wu Z,Shang Gdoi
10.1007/s00253-019-09744-9subject
Has Abstractpub_date
2019-04-01 00:00:00pages
3559-3570issue
8eissn
0175-7598issn
1432-0614pii
10.1007/s00253-019-09744-9journal_volume
103pub_type
杂志文章abstract：:Three bacterial strains utilizing paracetamol as the sole carbon, nitrogen, and energy source were isolated from a paracetamol-degrading aerobic aggregate, and assigned to species of the genera Stenotrophomonas and Pseudomonas. The Stenotrophomonas species have not included any known paracetamol degraders until now. I...
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