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YLC-assembly: large DNA assembly via yeast life cycle
Bo He1,2, Yuan Ma1,2, Fangfang Tian1,2
1Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Nucleic Acids Research
|July 24, 2023
Summary
We developed a novel yeast life cycle (YLC)-assembly method for efficient in vivo DNA assembly. This technique simplifies large DNA construction for synthetic biology and genome engineering applications.
Area of Science:
- Synthetic biology
- Molecular biology
- Genomics
Background:
- Large DNA assembly is crucial for synthetic biology but current methods are inefficient.
- Scaling up genetic manipulation requires robust large DNA assembly techniques.
Purpose of the Study:
- To develop an efficient and easy-to-operate method for in vivo iterative assembly of large DNA molecules.
- To overcome the limitations of existing cumbersome and inefficient large DNA assembly technologies.
Main Methods:
- Developed a yeast life cycle (YLC)-assembly method utilizing yeast mating and sporulation cycles for iterative DNA assembly.
- Employed nesting cell-cell transfer of assembled DNA within the yeast life cycle.
- Successfully assembled 100-kilobase (kb) endogenous yeast DNA and megabase (Mb)-sized exogenous DNA.
Main Results:
- Achieved over 10^4 positive colonies per 10^7 cells in each assembly round.
- Demonstrated high accuracy rates ranging from 67% to 100% for the assembled DNA.
- The YLC-assembly method showed a higher success rate compared to other Mb-sized DNA assembly techniques.
Conclusions:
- YLC-assembly provides a simplified workflow, avoiding difficult in vitro operations for large DNA.
- This method significantly lowers the technical barrier for megabase-sized DNA assembly.
- YLC-assembly is a valuable tool for large-scale genome engineering and advancing synthetic genomics.
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