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Acta Biochim Biophys Sin (Shanghai). 2019 Jan 1;51(1):97-103. doi: 10.1093/abbs/gmy139.

iCatch: a new strategy for capturing large DNA fragments using homing endonucleases.

Wang J1,2, Lu A1,2, Liu J3, Huang W1,4, Wang J5,6, Cai Z1,4, Zhao G1,5,6.

Author information

1
Carson International Cancer Center, Shenzhen University School of Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China.
2
State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China.
3
Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
4
Guangdong Key Laboratory of Systems Biology and Synthetic Biology, Urogenital Tumors, Shenzhen, China.
5
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
6
Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China.

Abstract

Natural genetic materials contain many biosynthetic gene clusters encoding potentially valuable natural products, many of which can be used directly without codon optimization or other manipulations. With the development of synthetic biology, several DNA assembly standards have been proposed, conveniently facilitating the reuse of natural materials. Among these standards, the iBrick assembly standard was developed by our laboratory to manipulate large DNA fragments, employing two homing endonucleases. Considering the difficulty of cloning large iBrick parts using conventional endonuclease-mediated restriction and ligation methods, we herein present a new method, known as iCatch, which readily captures biosynthetic gene clusters. As the clusters cloned by iCatch have the prefix and suffix of the iBrick standard, they serve as new iBrick parts and are therefore conducive to further editing and assembly with the iBrick standard. iCatch employs the natural homologous recombination system to flank the region of interest with I-SceI and PI-PspI recognition sites, after which the genome is digested with I-SceI or PI-PspI and the fragments are then self-ligated to clone the target DNA fragments. We used this method to successfully capture the actinorhodin biosynthetic cluster from Streptomyces coelicolor and then heterologously expressed this cluster in a thermophilic Streptomyces strain. We propose that iCatch can be used for the cloning of DNA sequences that are dozens of kilobases in length, facilitating the heterologous expression of microbial natural products. Moreover, this cloning methodology can be a complementary tool for the iBrick standard, especially in applications requiring the manipulation of large DNA fragments.

PMID:
30452545
DOI:
10.1093/abbs/gmy139
[Indexed for MEDLINE]

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