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Published on: August 12, 2019
Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid
Bogumil J Karas1, Christian Tagwerker, Isaac T Yonemoto
1J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121, USA. bkaras@jcvi.org
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.
Insights
Researchers successfully cloned the Acholeplasma laidlawii genome in yeast, a bacterium using the standard genetic code. They inactivated a toxic gene to enable stable cloning, advancing synthetic biology and genome engineering capabilities.
Area of Science:
- Synthetic biology
- Microbial genomics
- Yeast-based cloning technologies
Background:
- Cloning bacterial genomes in yeast is crucial for synthetic cell development.
- Previous work focused on Mycoplasma mycoides, which uses a non-universal genetic code.
- Expanding yeast cloning to bacteria with the universal genetic code presents new challenges.
Purpose of the Study:
- To clone the Acholeplasma laidlawii genome, which uses the universal genetic code, in yeast.
- To identify and overcome potential toxicity issues when cloning bacterial genes in a yeast host.
- To establish a method for stable maintenance of bacterial genomes as plasmids in yeast.
Main Methods:
- Whole genome cloning of Acholeplasma laidlawii PG-8A in Saccharomyces cerevisiae.
- Identification of toxic genes within the A. laidlawii genome through functional screening.
- Gene inactivation strategy to mitigate toxicity and ensure stable plasmid maintenance.
Main Results:
- Successful cloning of the A. laidlawii genome in yeast.
- Identified a single toxic gene: an extracellular endonuclease.
- Inactivation of the toxic gene allowed for stable cloning and maintenance of the A. laidlawii genome as a centromeric plasmid.
Conclusions:
- Yeast is a viable host for cloning bacterial genomes that utilize the universal genetic code.
- Targeted gene inactivation is an effective strategy to overcome toxicity in heterologous hosts.
- This work expands the toolkit for synthetic biology and large-scale genome engineering.
