Whole Genome Rebooting Paper Review
The paper “Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast” by Lartigue et al provides a method of reproducing a whole genome of a cloned bacterial cell (Mycoplasma mycoides) using yeast and subsequently transplanting the whole genome in another bacterial cell (Mycoplasma capricolum) to create a viable cell. A previous paper by the authors describes how the genome transplanted directly from one bacteria cell to another, while this paper focuses on its reproduction in yeast and then transplantation into another bacterial cell. There is not an overarching scientific experiment in this paper, as it is primarily a methods paper. The main idea of the paper is to show how they manipulated the plasmid containing the reproduced genome in order to allow it to be transplanted into another cell.
In order to reproduce the genome in yeast, the authors had to create a knockout cassette. They showed in Figure 1a a knockout of the Type III restriction endonuclease gene and replaced it with a cassette containing a URA3 marker and a Gal1 promoter paired with a SCEI endonuclease gene. This produced a seamless deletion of the Type III restriction endonuclease gene with the knockout cassette containing the URA3 marker. Further exposure to 5-fluoroorotic acid produced a similar deletion that did not contain the URA3 marker. PCR and gel electrophoresis in Figure 1b showed that in both yeast and post-transplant genome the knockout cassette was present with the fragment sizes that were expected for both the cassette containing URA3 and without URA3.
One of the main problems that the authors had in transplanting the genome into the recipient cell was that a restriction endonuclease was interfering and degrading the genome before transplantation could occur. They solved this problem using two methods. The first method entailed modifying the recipient Mycoplasma capricolum cell in order to inactivate the restriction enzyme by placing a resistance marker in the coding region of the gene. The second method was to use in vitro methylation of the donor genome in order to keep the restriction enzyme from affecting the genome. Testing revealed that both methods were effective in keeping the recipient cell’s restriction enzymes from degrading the donor DNA.
In order to increase the viability of the method, the authors next tested two different yeast strains as a host for the reproduction and transplantation of the genome. Also using different methylation techniques, Table 1 shows that the genome can successfully be transplanted to recipient bacterial cells for both yeast strains.
Once the genome was transplanted into the recipient cell, they had to be sure that the transplantation occurred successfully. In Figure 2a, they used Southern blot analysis using the IS1296 marker, which only occurs in Mycoplasma mycoides. Comparison of native and transplanted Mycoplasma mycoides and native Mycoplasma capricolum genomes show that the former all contained the IS1296 marker sequence. In Figure 2b, they also use a Southern blot, but instead use a probe for a sequence within the deleted region of the genome (the part that was deleted by the knockout cassette). The results showed that the native Mycoplasma mycoides contained the sequence, while none of the transplant genomes contained the sequence, providing a simple way of determining whether or not the genome was successfully transplanted or not. At this point, the authors have successfully completed the reproduction of the bacterial genome in yeast and the transplantation of the genome in another bacterial cell.
Figure 3 provides a simplified view of the entire process of whole genome rebooting. First, a yeast vector is inserted into the bacterial genome to allow the vector to be transformed into a yeast cell. Next, the genome (while in the yeast cell) can be engineered and then isolated. Then, the methylation of the genome is done before transplantation into the recipient cell. Finally, screening and Southern blot analysis can be used to determine whether the genome was transplanted successfully or not.
Now that the method of reproducing and transplanting the bacterial genome is complete, the question of why this was done needs to be answered. The transfer of the genome to yeast allows the vast resources and genetic techniques available to yeast research to be performed on a bacterial genome. Most bacteria (including the Mycoplasma family of bacteria) are limited in the genetic tools available to them. This opens the possibility of future research of working with bacteria that are difficult to manipulate using the powerful genetic tools available to yeast. The possibilities of what can be done are quite wide, as now any bacteria that can be successfully processed in this way becomes a simplified research bacteria that can be manipulated and engineered in a relatively simple way. Now new research into troublesome bacteria such as those that cause diseases in humans and other animals can be explored in order to possibly come up with new treatments or cures for various diseases. The authors cite at the very end of the paper that the Mycoplasma family of bacteria are responsible for major diseases in ruminants such as cattle, so further research into this bacteria could have potential treatments or cures for these diseases in the future. This is a very powerful method that the authors have discovered, and the future possibilities could have just as much of an effect.
Reference
Lartigue, Carole, Sanjay Vashee, Mikkel A. Algire, Ray-Yuan Chuang, Gwynedd A. Benders, Li Ma, Vladimir N. Noskov, Engeniya A. Denisova, Daniel G. Gibson, Nacyra Assad-Garcia, Nina Alperovich, David W. Thomas, Chuck Merryman, Clyde A. Hutchinson III, Hamilton O. Smith, J. Craig Venter, and John I Glass. 2009. “Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast.” Science, Vol 325, pp. 1693-1696.
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