A CRISPR-based chromosomal-separation technique for Escherichia coli

Autor: Junchang Su, Pengju Wang, Ju Li, Dongdong Zhao, Siwei Li, Feiyu Fan, Zhubo Dai, Xiaoping Liao, Zhitao Mao, Chunzhi Zhang, Changhao Bi, Xueli Zhang
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Microbial Cell Factories, Vol 21, Iss 1, Pp 1-11 (2022)
Druh dokumentu: article
ISSN: 1475-2859
DOI: 10.1186/s12934-022-01957-4
Popis: Abstract Background Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains. Results The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli 0.10/4.54 and E. coli 2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli 0.10/4.54 was quite stable, the two chromosomes of E. coli 2.28/2.36 population recombined into a new chromosome (Chr.4.64MMut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated. Conclusion Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.
Databáze: Directory of Open Access Journals
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