Dataset Information

A CRISPR-based chromosomal-separation technique for Escherichia coli.

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.4.64M^Mut), 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.

SUBMITTER: Su J

PROVIDER: S-EPMC9652834 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Publications

A CRISPR-based chromosomal-separation technique for Escherichia coli.

Su Junchang J Wang Pengju P Li Ju J Zhao Dongdong D Li Siwei S Fan Feiyu F Dai Zhubo Z Liao Xiaoping X Mao Zhitao Z Zhang Chunzhi C Bi Changhao C Zhang Xueli X

Microbial cell factories 20221111 1

<h4>Background</h4>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.<h4>Results</h4>The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was deve ...[more]

PMID: 36369085

Similar Datasets

Project description:UnlabelledClustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes constitute the CRISPR-Cas systems found in the Bacteria and Archaea domains. At least in some strains they provide an efficient barrier against transmissible genetic elements such as plasmids and viruses. Two CRISPR-Cas systems have been identified in Escherichia coli, pertaining to subtypes I-E (cas-E genes) and I-F (cas-F genes), respectively. In order to unveil the evolutionary dynamics of such systems, we analyzed the sequence variations in the CRISPR-Cas loci of a collection of 131 E. coli strains. Our results show that the strain grouping inferred from these CRISPR data slightly differs from the phylogeny of the species, suggesting the occurrence of recombinational events between CRISPR arrays. Moreover, we determined that the primary cas-E genes of E. coli were altogether replaced with a substantially different variant in a minor group of strains that include K-12. Insertion elements play an important role in this variability. This result underlines the interchange capacity of CRISPR-Cas constituents and hints that at least some functional aspects documented for the K-12 system may not apply to the vast majority of E. coli strains.ImportanceEscherichia coli is a model microorganism for the study of diverse aspects such as microbial evolution and is a component of the human gut flora that may have a direct impact in everyday life. This work was undertaken with the purpose of elucidating the evolutionary pathways that have led to the present situation of its significantly different CRISPR-Cas subtypes (I-E and I-F) in several strains of E. coli. In doing so, this information offers a novel and wider understanding of the variety and relevance of these regions within the species. Therefore, this knowledge may provide clues helping researchers better understand these systems for typing purposes and make predictions of their behavior in strains that, depending on their particular genetic dotation, would result in different levels of immunity to foreign genetic elements.

Dataset Information

A CRISPR-based chromosomal-separation technique for Escherichia coli.

Background

Results

Conclusion

Publications

A CRISPR-based chromosomal-separation technique for Escherichia coli.

Similar Datasets

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