DNA assembly for free: using cellular lysates to decrease cloning costs

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Adam Fisher

Virginia Commonwealth University, United States

Recent advancements in DNA synthesis and assembly have continued to drive forward synthetic biology research. However, DNA assembly techniques such as isothermal DNA assembly and transformation-associated recombination (TAR) cloning, are expensive in terms of time or money. Here we propose to investigate the capabilities of lysates from wild-type Escherichia coli, Saccharomyces cerevisiae and Deinococcus radiodurans to mediate DNA assembly. The process is as simple as culturing and harvesting lysate from your assembly microbe, adding linear DNA fragments, incubating for an hour and directly transforming circularized products. This assembly methodology harnesses the same innate DNA-repair mechanisms exploited in TAR cloning, yet obviates the need for transformation and subsequent isolation of S. cerevisiae-compatible final products. With respect to Gibson’s Isothermal assembly, this process is significantly less expensive per reaction, improving assembly throughput with limited resources. We expect this lysate-based DNA assembly approach to further enable the implementation of genetically encoded systems of increasing size and complexity for both applications and basic biological research.