New technologies to synthesize DNA provides a great opportunity to completely redesign the entire genome of an organism. Teamed with several research groups worldwide, we aim to re-synthesize a designer eukaryotic genome, Sc2.0. In my lab, a 976,067-base pair linear chromosome, synXII, was designed and assembled using a two-step method, producing a functional chromosome. The ribosomal gene cluster (rDNA) on synXII is retained during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, commonly used as the molecule barcode of a species, are swapped to generate a Saccharomyces strain that would be identified as Saccharomyces bayanus. Furthermore, as a novel inducible system implemented in the synthetic chromosomes, SCRaMbLE is designed to generate diverse genotypes and phenotypes by massive chromosome rearrangements. We have designed a reporter of SCRaMbLEd cells using efficient selection, termed ReSCuES, based on a loxP-mediated switch of two auxotrophic markers. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Using ReSCuES, we illustrate the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. In addition, by analyzing the tolerant strains, we are able to identify ACE2, a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance. Collectively, our work not only offers a new avenue of decoding the yeast genome through intelligent design followed by chemical synthesis, but also demonstrates our ability to reprogram the genome for future applications.
Dr. Dai is a Professor at the Shenzhen Institutes of Advanced Technology (SIAT) and also Director of the Center for Synethic Genomics at SIAT. The Dai Lab uses budding yeast as model organism to study the function of chromatin with emphasis on histone modifications. In addition, they are synthesizing the yeast chromosome XII as a part of the international collaborative project (the Sc2.0 project) and developing technologies for synthetic biology. Dr. Dai has published over 20 papers in major scientific journals and has been awarded several patents.
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