Molecular cloning is usually carried out on one gene or small DNA segment at a time. However, cloning technology has advanced to the stage that scientists have begun cloning genomes of entire unicellular organisms. This approach could be valuable for engineering organisms that thus far have not been amenable to propagation and engineering through conventional laboratory methods.
Cloning those organism’s genomes into a host system would allow the genome to be manipulated in that system for research. This type of cloning experiment has been carried out successfully for a number of different unicellular organisms.
Protozoons / unicellular organisms - Image Credit: Christoph Burgstedt / Shutterstock
Bacterial Genomes Cloned into Yeast
In one study, published in 2010, investigators describe the cloning of the genomes of Mycoplasma genitalium, M. pneumoniae, and M. mycoides in the yeast Saccharomyces cerevisiae. The genomes were cloned using a yeast centromere, a circular form of DNA native to yeast.
In order to be propagated in yeast, the bacterial genome must include some yeast sequences. Those are incorporated by standard molecular cloning in the bacterial genome. They can also be added by co-transformation in the yeast. Another option is cloning by assembly of multiple, overlapping fragments.
A significant fraction of yeast transformants prepared by these methods had complete mycoplasma genomes. Some clones contained incomplete mycoplasma genomes, for unknown reasons. Sequences of M. mycoides genomes isolated from the cloned yeast showed the same sequence as the parent organism, except for changes made for the purposes of cloning.
These methods may be useful for studying organisms that don’t grow under laboratory conditions or do not have readily available genetic tools. For example, pathogens and organisms collected by environmental sampling. Once the genome is cloned in yeast, it can be manipulated with standard genetic engineering methods for yeast.
Another application would be genome transplantation, where an engineered genome is installed in a new bacterial cell and manipulated to create an organism with certain traits.
In a 2007 publication, researchers describe a genome transplantation experiment. They transplanted intact genomic DNA from Mycoplasma mycoides large colony into M. capricolum cells using polyethylene glycol-mediated transformation. The resulting cell line contained the complete donor genome, free of recipient genomic sequences and were phenotypically identical to the donor strain.
According to the J. Craig Venter Institute, cloning whole bacterial genomes from one unicellular organism to another is a step toward creating a synthetic genome--a major goal of the institute. Scientists at JCVI followed up on the 2007 bacterial transplantation experiment by cloning a whole bacterial genome from M. mycoides into a yeast cell. They then isolated the cloned bacterial chromosome and transplanted it into a related bacterium, M. capricolum, creating a new type of M. mycoides.
It was the first time that a genome had been transferred from a prokaryote to a eukaryote and back to a prokaryote again. One of the challenges of the final stage was methylating the cloned genome from yeast so that it could be transformed back into a bacterium.
- Creation of a bacterial cell controlled by a chemically synthesized genome, https://www.ncbi.nlm.nih.gov/pubmed/20488990
- Genome transplantation in bacteria: changing one species to another, https://www.ncbi.nlm.nih.gov/pubmed/17600181
- J. Craig Venter Institute researchers clone and engineer bacterial genomes in yeast and transplant genomes back into bacterial cells, https://www.jcvi.org/j-craig-venter-institute-researchers-clone-and-engineer-bacterial-genomes-yeast-and-transplant
- Cloning whole bacterial genomes in yeast, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860123/