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Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System
Published on: August 17, 2017
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Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides
Huiran Yeom1, Taehoon Ryu2, Amos Chungwon Lee3
1Department of Electrical and Computer Engineering, Seoul National University, Seoul, 08826, South Korea.
ACS Synthetic Biology
|May 9, 2020
Summary
Synthesizing engineered bacteriophages (phages) is crucial for applications like phage therapy. A new cell-free Sniper assembly method enables low-cost, efficient de novo phage genome construction using synthesized DNA.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Biotechnology
Background:
- Engineered bacteriophages (phages) offer potential in drug screening and phage therapy.
- Conventional phage engineering relies on inefficient in vivo systems with low production and transformation efficiency.
Purpose of the Study:
- To develop a cell-free, cost-effective de novo gene synthesis technology for phage genome construction.
- To overcome the limitations of traditional in vivo phage engineering methods.
Main Methods:
- Utilized Sniper assembly, a cell-free technology for de novo phage genome construction.
- Employed massively parallel sequencing of microarray-synthesized oligonucleotides (oligos).
- Generated and identified approximately 100,000 clonal DNA clusters in vitro and 5,000 error-free clones in a cell-free environment.
Main Results:
- Successfully synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones.
- Achieved a significantly reduced genome synthesis cost of $0.0137/bp.
- Demonstrated the production of low-cost, sequence-verified DNA for phage genome construction.
Conclusions:
- Sniper assembly provides a viable cell-free approach for efficient and economical de novo phage genome synthesis.
- This technology enhances the potential for engineering phages for diverse applications, including therapeutic uses.
- The low cost and high efficiency position Sniper assembly as a valuable tool in synthetic biology and phage research.
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