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.



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.
iii.  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.