Karas Bogumil J, Tagwerker Christian, Yonemoto Isaac T, Hutchison Clyde A, Smith Hamilton O
J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121, USA.
ACS Synth Biol. 2012 Jan 20;1(1):22-8. doi: 10.1021/sb200013j. Epub 2012 Jan 10.
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.
在酵母中克隆支原体属的全基因组是创建首个合成细胞的关键步骤。合成细胞的基因组基于蕈状支原体，它偏离了通用遗传密码，通过编码色氨酸而非UGA终止密码子。这一特性被认为很重要，因为如果由在酵母中活跃的隐秘启动子驱动，细菌基因可能对宿主酵母细胞有毒性。随着我们着手扩大在酵母中克隆的细菌基因组范围，我们将这项技术扩展到了使用通用遗传密码的细菌。在此我们报告了莱氏无胆甾原体PG - 8A基因组的克隆，该细菌使用通用遗传密码。我们发现，当在酵母中克隆时，只有一个莱氏无胆甾原体基因，即一种表面锚定的细胞外核酸内切酶，具有毒性。为了在酵母细胞中作为着丝粒质粒克隆并稳定维持莱氏无胆甾原体基因组，该基因被失活。
