Overview

In this paper, the authors describe a novel research method in which the genome of a prokaryotic cell may be modified via introduction into a eukaryotic cell. By isolating the genome of the baterium Mycoplasma mycoides into the eukaryotic yeast strains VL6-48N and W303a, the prokaryotic genome may be modified using any known yeast genetic tools and methods to generate any genetic changes researchers would like to make. The modified genome is then transplanted back into a prokaryotic cell of the bacterium Mycoplasma capricolum, which then produced M. mycoides strains containing all the modifications that were made. This method allows researchers to alter the genome of the M. mycoides bacterium using genetic tools that could not be used directly on the M. mycoides bacterium. 

Results

Figure 1. 

Part A of this figure depicts the changes that were made to the M. mycoides bacterium genome. A vector was introduced to the M. mycoides genome at the site of a nonessential Type III restriction endonuclease gene, effectively replacing that gene with a vector containing a selectable tetracyline-resistance marker as well as a β-galactosidase gene to be used for screening, a yeast auxotrophic marker, and a yeast centromeric plasmid which is used for selection and propagation in yeast. Collectively the M. mycoides genome and the yeast vector were referred to as the YCpMmyc1.1. In part (i), the normal YCpMmyc1.1 gene was shown, including the Type III restriction enzyme gene (typeIIIres) that will be replaced by a vector containing  a URA3 gene, which is a marker, and an SCEI endonuclease gene, which is downstream of a GAL1 promoter. This entire vector included a tandem repeat sequence and was referred to as the knockout cassette. The cassette was then introduced into the W303a yeast strain that already includes the YCoMmyc1.1 M. mycoides genome; this process was done by growing those yeast cells in an environment lacking His and Ura, which allowed the cassette to enter and replaces the Type III restriction enzyme as seen in (ii). Part (iii) depicts a YCoMmyc1.1 M. mycoides genome lacking both the typeIIIres and a yeast vector, which will be used as a point of comparison for Part B of this figure. Due to the galactose induction and 5-fluoroorotic acid counter selection, it was possible to generate construct (iii) as well as construct (ii). The arrows that are seen in all the parts of this figure are different PCR primers that will be used to determine whether the cassette has been successfully incorporated into the YCpMmyc 1.1.

Part B depicts a DNA blot that indicates the presence of various constructs that were shown in Part A. PCRs of the various constructs were shown both in the yeast cell and after transplant of the cassette has taken place. In both cases, we can see that construct (ii) was heavier and moved more slowly due to the higher amount of bases it carried, whereas construct (iii) is significantly lighter than constructs (i) and (ii), since it has neither the typeIIIres nor the yeast vector. Interestingly, construct (i) in post-transplant has a brighter signal than construct (i) in yeast, which may indicate a higher amount of that construct in post-transplant. Overall, it seems that the typeIIIres was indeed successfully deleted from the YCoMmyc1.1 genome and replaced by the heavier cassette. 

Table 1.

The researchers discovered that transplantation of YCpMmyc 1.1 genomes from the yeast strains into the recipient M. capricolum bacterial cell did not generate any implants. To explore this problem, they inactivated the single restriction enzyme in M. capricolum and sought to protect the incoming YCpMmyc 1.1 genomes via in vivo methylation using both M. mycoides and M. capricolum “extracts,” though it were not clear what the extracts consisted of. This Table shows the various constructs of the YCpMmyc 1.1 genome, the methylation status, and the number of colonies that were seen of both wild type M. capricolum and M. capricolum RE(-), whose restriction enzyme has been deactivated. “Untreated” indicates that the cells were digested with β-agarase (called the melting step) prior to being transplanted to both types of M. capricolum cells. “Treated” indicates that samples were methylated and then transplanted. “Mock-treated” indicates that the samples were treated similarly to “treated” samples, but no extract of purified methyltransferases were added. This results show that untreated and mock-treated YCpMmyc 1.1 were not able to be transplanted in wild-type M. capricolum, although they were successfully transplanted in M. capricolum RE(-).YCpMmyc 1.1 which were methylated with extracts from either M. capricolum or M. mycoides were successfully transplanted in both types of M. capricolum, indicating that the M. capricolum  restriction enzyme was the factor preventing successful transplanting and that methylation of the YCpMmyc 1.1 can overcome this problem. One of the samples, a YCpMmyc 1.1 that lacks 500 kilobases, served as a negative control, since it is known to lack many presumably essential genes and thus would not have been transplanted by any type of M. capricolum cells. These results also show that the YCpMmyc 1.1 genome can be grown in both the VL6-48N and the W303a yeast strains.

Figure 2. 

These figures were conducted with the aim of proving that the colonies contained only M. mycoides DNA and not any other kind of DNA sequences of combinations thereof.

(A) shows a Southern blot analysis of M. mycoides transplants, which used a M. mycoides-specific IS1296 element as a probe. These genes were also Hind III restricted. The wild-type M. capricolum lane was meant to be used as a negative control, and the native M. mycoides YCpMmyc 1.1 was used as a positive control. The other lanes indicate the various constructs seen in Figure 1. These constructs shared the same detected banding patterns as M. mycoides YCpMmyc 1.1, which suggests that they are M. mycoides DNA. The inclusion of the construct in the last lane, typeIIIres, is important since it ruled out the possibility that yeast DNA was detected (since this construct has no yeast DNA, yet has the same banding patterns as M. mycoidesYCpMmyc1.1).

(B) shows a Southern blot analysis using the typeIIIres gene as probe. The samples tested were the same as those in (A), except that transplant M. mycoides YCpMmyc 1.1 was not included. The blot shows a band only in the native M. mycoides YCpMmyc 1.1, which was used as the positive control, and not in any of the engineered genomes. The wild type M. capricolum was used as a negative control.

(C) shows the DNA sequence of the locus where the typeIIIres was presumably deleted. The start and stop codons of this gene is boxed in red, whereas the stop codon of the typeIIImod gene is boxed in black. The typeIIImod black stop codon lie between the two red codons. A small DNA sequence of where the typeIIIres was is seen, but that sequence was argued to be because of the overlap between the typeIIIres and the typeIIImod.

Figure 3. 

This figure summarizes the technique that the authors described. The genome of a bacterium is transformed by means of a yeast vector, allowing this genome to then be cloned into a yeast cell. At this point, the genome may be altered using any known yeast genetic modification methods, allowing the possibility of creating new genomes. The newly altered genome then is then able to be transplanted into a recipient bacterium cell, which would make more copies of the altered genome during replication. Note that methylation of the newly altered genome may be necessary to prevent the breakdown of this genome upon reintroduction into a bacterium cell. This transplant of a prokaryotic genome into a eukaryotic cell allows researchers to adjust the genome using methods that would not be possible in a prokaryotic cell. The dashed arrow indicates that the newly altered genome may be isolated and undergo this cycle again so that researchers can further modify the genome as they see fit.

Critique

Over all I found the evidence presented to be quite compelling and interesting. The researchers have taken great care to show that bacterial genome can be imported into a yeast, and then from the yeast back into a different type of bacterial cell. I found that their evidence strongly supported their claims, and overall I found no glaring, obviously problematic weakness or doubtful evidence.

I wonder why the Southern blots displayed in Figure 2 were not all one blot, but were multiple juxtaposed pieces. Having all of those lanes on one blot would have left less room for doubt, and certainly more aesthetically pleasing. Also, I would have liked to see the transplant M. mycoides YCpMmyc 1.1 included in Figure 2 (B) for the sake of consistency and complete comparison. I also would have liked to see some mention of signaling or loading equality, just to ensure that there isn’t an amount of typeIIIres present but just at low and undetected quality. I also felt as though  the argument the authors attempted to make in Figure 2 (C) could have been presented more powerfully.

For Table 1, I would have liked to see the researchers attempted transplant YCpMmyc 1.1 grown in the W303a yeast strain to the wild-type M. capricolum. Even if they had strong reasons to believe that it might not have happened and thus decided not to conduct that action, I believe that their argument would have been more complete and secure if they had attempted such a transplanting and displayed the results.

Future Directions

When discovering such a novel tool, I believe that it ought to be utilized as much as possible to discover any weaknesses. This technique should be practiced such that various combinations of genetic changes should be conducted in order to discover any inherent limitations within the technique that hitherto has not been seen. Many patterns of manipulations should be conducted and carefully examined if all of the alterations that were made are seen in the final engineered form. To this end I would continue to practice the method repeatedly.