Cloning the Acholeplasma laidlawii PG-8A Genome in Saccharomyces cerevisiae as a Yeast Centromeric Plasmid
Cloning the Acholeplasma laidlawii PG-8A Genome in Saccharomyces cerevisiae as a Yeast Centromeric Plasmid
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DOI：
10.1021/sb200013j
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发表时间：
2012-01-01
影响因子：
4.7
通讯作者：
Smith, Hamilton O.
中科院分区：
文献类型：
--
作者：
Karas, Bogumil J.;Tagwerker, Christian;Smith, Hamilton O.
在酵母中克隆支原体属的全基因组是创造第一个合成细胞的关键步骤。合成细胞的基因组基于丝状支原体，其通过编码色氨酸而不是UGA终止密码子而偏离通用遗传密码。这一特征被认为是重要的，因为如果细菌基因由酵母中活跃的隐蔽启动子驱动，那么细菌基因可能对宿主酵母细胞有毒。随着我们扩大在酵母中克隆的细菌基因组的范围，我们将这项技术扩展到使用通用遗传密码的细菌。在这里，我们报告的莱氏无胆甾原体PG-8A基因组的克隆，它使用的通用遗传密码。我们发现只有一个A. Laidlawii基因是一种表面锚定的胞外核酸内切酶，当克隆到酵母中时具有毒性。为了克隆和稳定保存A. Laidlawii基因组作为着丝粒质粒存在于酵母细胞中。
Cloning of whole genomes of the genus Mycoplasma in yeast has been an essential step for the creation of the first synthetic cell. The genome of the synthetic cell is based on Mycoplasma mycoides, which deviates from the universal genetic code by encoding tryptophan rather than the UGA stop codon. The feature was thought to be important because bacterial genes might be toxic to the host yeast cell if driven by a cryptic promoter active in yeast. As we move to expand the range of bacterial genomes cloned in yeast, we extended this technology to bacteria that use the universal genetic code. Here we report cloning of the Acholeplasma laidlawii PG-8A genome, which uses the universal genetic code. We discovered that only one A. laidlawii gene, a surface anchored extracellular endonuclease, was toxic when cloned in yeast. This gene was inactivated in order to clone and stably maintain the A. laidlawii genome as a centromeric plasmid in the yeast cell.