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
Description of Data
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.
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.
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.
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
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.
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
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
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.
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