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 inconv...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-11-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12934-022-01957-4 |
| _version_ | 1811225072517513216 |
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| author | Junchang Su Pengju Wang Ju Li Dongdong Zhao Siwei Li Feiyu Fan Zhubo Dai Xiaoping Liao Zhitao Mao Chunzhi Zhang Changhao Bi Xueli Zhang |
| author_facet | Junchang Su Pengju Wang Ju Li Dongdong Zhao Siwei Li Feiyu Fan Zhubo Dai Xiaoping Liao Zhitao Mao Chunzhi Zhang Changhao Bi Xueli Zhang |
| author_sort | Junchang Su |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T08:59:27Z |
| format | Article |
| id | doaj.art-7f67eeb6c6344612bd842ae15295c01e |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-12T08:59:27Z |
| publishDate | 2022-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-7f67eeb6c6344612bd842ae15295c01e2022-12-22T03:39:16ZengBMCMicrobial Cell Factories1475-28592022-11-0121111110.1186/s12934-022-01957-4A CRISPR-based chromosomal-separation technique for Escherichia coliJunchang Su0Pengju Wang1Ju Li2Dongdong Zhao3Siwei Li4Feiyu Fan5Zhubo Dai6Xiaoping Liao7Zhitao Mao8Chunzhi Zhang9Changhao Bi10Xueli Zhang11School of Biological Engineering, Dalian Polytechnic UniversityTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesCollege of Life Science, Tianjin Normal UniversityTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesSchool of Biological Engineering, Dalian Polytechnic UniversityTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesAbstract 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.https://doi.org/10.1186/s12934-022-01957-4Synthetic biologyGenome engineeringChromosomal-separationCRISPR-Cas9 |
| spellingShingle | Junchang Su Pengju Wang Ju Li Dongdong Zhao Siwei Li Feiyu Fan Zhubo Dai Xiaoping Liao Zhitao Mao Chunzhi Zhang Changhao Bi Xueli Zhang A CRISPR-based chromosomal-separation technique for Escherichia coli Microbial Cell Factories Synthetic biology Genome engineering Chromosomal-separation CRISPR-Cas9 |
| title | A CRISPR-based chromosomal-separation technique for Escherichia coli |
| title_full | A CRISPR-based chromosomal-separation technique for Escherichia coli |
| title_fullStr | A CRISPR-based chromosomal-separation technique for Escherichia coli |
| title_full_unstemmed | A CRISPR-based chromosomal-separation technique for Escherichia coli |
| title_short | A CRISPR-based chromosomal-separation technique for Escherichia coli |
| title_sort | crispr based chromosomal separation technique for escherichia coli |
| topic | Synthetic biology Genome engineering Chromosomal-separation CRISPR-Cas9 |
| url | https://doi.org/10.1186/s12934-022-01957-4 |
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