title: Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid.
abstract: 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.
authors: J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121, USA. bkaras@jcvi.org
authors: Bogumil J
authors: BJ
authors: Karas
authors: Christian
authors: C
authors: Tagwerker
authors: Isaac T
authors: IT
authors: Yonemoto
authors: Clyde A
authors: CA
authors: Hutchison
authors: Hamilton O
authors: HO
authors: Smith
mesh_terms: D000128:Acholeplasma laidlawii / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D002503:Centromere / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D005818:Genetic Engineering; D016680:Genome, Bacterial; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology
chemical_list: D001426:Bacterial Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; C038485:GGGCCC-specific type II deoxyribonucleases