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Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast

Summary:  The paper's main goal was to show that the researchers had successfully demonstrated that novel bacterial strains of Mycoplasma mycoides could be created by transforming the bacterial genome into yeast and using yeast-specific genetic engineering techniques before transplanting the genome back into bacteria.

Description of Data

Figure 1A:
 This figure is a pictorial demonstration of the genetic engineering performed, including the selection conditions, sizes of each, and the PCR primers used in Figure 1B, along the way.
i. YCpMmyc1.1: This diagram shows the unengineered genome as it would be just after transformation into the yeast cells, and the engineered DNA fragment (cassette) that is going to insert into the genome.  The typeIIIres gene is the gene being deleted.  Gal1P is the researcher-activated promoter.  SCEI is a gene coding for a restriction enzyme that cuts at the asterisk.  URA3 is a yeast selection marker, which is used to identify the yeast that successfully inserted the fragment.  TR is a section of tandem repeats that will later be used to remove the insert before genome transplantation back into bacteria.
ii.  YCpMmyc1.1-ΔtypeIIIres::URA3:  This is the resultant plasmid after insertion of the cassette.  Only yeast containing this plasmid should have been able to grow on the limited medium used.
YCpMmyc1.1-ΔtypeIIIres:  The final engineered form of the genome after removal of the cassette.  The introduction of galactose activated the Gal1 promoter, which promoted the transcription and translation of the SCEI gene and subsequent cutting by the restriction enzyme at the asterisk.  Homologous recombination between the two homologous tandem repeat areas should result in the loss of most of what was the cassette.  Selection for the yeast that performed this recombination was done by the 5-FOA, which should kill any yeast still containing URA3.
Figure 1B: This is a gel showing bands of the expected size at each step along the engineering pathway, and showing that the genome is stable through transplantation, as the fragment sizes are the same in yeast and in post-transplantation bacteria.  This serves as confirmation that the expected insertions and deletions actually took place.

Table 1: This table shows that conditions needed for successful transplantation back into bacteria.  After unsuccessful transplantation of the genome into wild type bacteria, the researchers hypothesized that a restriction enzyme in the bacteria was degrading the donor genome.  To get around this the researchers made a strain of bacteria in which the single restriction enzyme gene was disrupted and/or they methylated the donor DNA in a series of ways.  Bacterial colonies grew when genomes of every condition were transplanted into the restriction-altered strain, except for the negative control (
YCpMmyc1.1-Δ500kb, which had essential genes deleted).  As long as the donor DNA was methylated, transplantation was successful into wild-type bacteria as well.

Figure 2A:  This is a Southern Blot showing that transplantation of an engineered M. mycoides genome into M. capricolum did make the resulting colonies into M. mycoides colonies.  The probe was a M. mycoides specific probe, so it shouldn't bind the wild type M. capricolum (lane 1), which it does not.  The band patterns also match between all the transplants and the native M. mycoides, as they should if they were the same species.
Figure 2B:  This is another Southern Blot that is probed with the typeIIIres sequence to check for the presence of the typeIIIres gene.  Referring back to Figure 1A, only the native M. mycoides YCpMmyc1.1 genome should have it, and that is the only one for which a band appears.
Figure 2C:  Sequencing of the
YCpMmyc1.1-ΔtypeIIIres transplant to verify that the typeIIIres gene was deleted as was intended.  There is a little of the gene left, but the majority of it was been deleted, leaving mostly the typeIIImod and IGR genes.


The results certainly support the writer's claims.  The argument is clearly and persuasively laid out, each point stemming from those before it and supported by the figures.  Though there were not any specific results presented in Figure 1A, it was extremely useful as a reference to follow what should be happening and whether the subsequent results supported those ideas.  Figure 1B is important in that it supports that the proposed insertions and deletions are actually occurring, and that the sequences are stable through the process of transplantation too.  From this fact that the engineering techniques are successful comes Table 1, showing that the engineered transplants are viable bacterial genomes despite being engineering in an entirely different domain.  After showing that the genome creates a viable bacterium the next question is which species the resultant bacterium is, which is answered by Figure 2A.  Figure 2B serves as yet another result showing that the yeast genome engineering is successful and maintained within the resulting transplanted bacteria.  Figure 2C is very specific and clear evidence that the newly engineered M, mycoides strain lacks the typeIIIres gene.  Sequencing is the strongest way to support that conclusion and that is what they have included.  Figure 3 could have easily been left out, but serves as a nice over-view of the method.

My only complaint is that the researchers did not include wild type M. mycoides in the Southern Blots of Figure 2A & B.  For 2A, this inclusion could have been a positive control, further confirming that the probe was M. mycoides specific and that it was not binding to something else the different lanes shared, such as a part of the original vector introduced into the bacterial genome.  Would have been nice to have the wild type M. mycoides serve as positive control in 2B as well, if only to provide more information and both a positive and negative control.

Possible Future Work

I would be interested in two questions that are slightly related: does the method work for other species and genera of bacteria, and can this be used to develop non-pathogenic bacterial strains for use as vaccines?  The first question first.  Mycoplasmas are among the smallest bacteria, some closer in size to viruses, so it would be interesting to see if the technology is applicable to the larger bacteria.  Since I would be testing how universal the method is, the method would likely stay the same.  I expect that the effectiveness would carry over, but I am worried about how well yeast would handle transformation of larger genomes as well as how stable the large genomes themselves would be through transformation and transplantation.

If this technology proves fairly universal, it could greatly increase our ability to produce vaccines, especially for bacteria like Mycoplasmas that are resistant to antibiotics like penicillin by virtue of not having a cell wall.  If we could knock out those genes responsible for a bacteria's pathogenicity and grow large amounts of the bacterium, it would both increase our supply of vaccines and make them more available/cheaper.  I would take a bacteria strain which is pathogenic in a model organism such as mice, but very well understood genetically, and look for pathogenic genes that are nonessential to knockout through this process.  By specifically knocking out these genes, the new strain should outwardly appear to be the wild type strain, serving as a vaccine against the pathogenic wild type.

Works Cited

Lartigue C, Vashee S, Algire M, Chuang R, Benders G, Ma L, Noskov V, Denisova E, Gibson D, Assad-Garcia N, Alperovich N, Thomas D, Merryman C, Hutchison C, Smith H, Venter J, Glass J.  Creating bacterial strains from genomes that have been cloned and engineered in yeast.  Science 325:  1693-1696.

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