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

Jingman Wang1,2, Anrui Lu1,2, Jiakun Liu3

  • 1Carson International Cancer Center, Shenzhen University School of Medicine, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China.

Acta Biochimica Et Biophysica Sinica
|November 20, 2018
PubMed
Summary

A new DNA cloning method, iCatch, efficiently captures large biosynthetic gene clusters using homologous recombination. This method facilitates the assembly and heterologous expression of natural products, complementing the iBrick standard for synthetic biology applications.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Genomics

Background:

  • Natural genetic materials harbor valuable biosynthetic gene clusters for natural products.
  • Synthetic biology relies on DNA assembly standards for reusing genetic materials.
  • The iBrick standard facilitates manipulation of large DNA fragments but faces cloning challenges.

Purpose of the Study:

  • To develop a novel method for efficiently cloning large biosynthetic gene clusters.
  • To create a method compatible with the iBrick DNA assembly standard.
  • To enable heterologous expression of microbial natural products.

Main Methods:

  • The iCatch method utilizes natural homologous recombination.
  • It flanks target gene clusters with specific endonuclease recognition sites (I-SceI and PI-PspI).
  • Genome digestion and self-ligation are employed to capture DNA fragments.

Main Results:

  • Successfully cloned the actinorhodin biosynthetic cluster from Streptomyces coelicolor.
  • Heterologously expressed the cloned cluster in a thermophilic Streptomyces strain.
  • Demonstrated iCatch's capability for cloning large DNA sequences (tens of kilobases).

Conclusions:

  • iCatch is an effective method for capturing large DNA fragments, particularly biosynthetic gene clusters.
  • The cloned fragments are compatible with the iBrick standard for further manipulation.
  • This method advances the heterologous expression of microbial natural products and complements large DNA fragment manipulation in synthetic biology.