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A Review of:
Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast
Lartigue, Sanjay Vashee, Mikkel A. Algire, Ray-Yuan Chuang, Gwynedd A.
Benders, Li Ma, Vladimir N. Noskov, Evgeniya A. Denisova, Daniel G.
Gibson, Nacyra Assad-Garcia, Nina Alperovich, David W. Thomas, Chuck
Merryman, Clyde A. Hutchison III, Hamilteon O. Smith, J. Craig Venter,
John I. Glass
As seen in Science, Vol. 325: 1693-1696. 25 September 2009
A Brief Summary
Lartigue et al. present a method for
modifying genomes outside their host organism. After removing the
genome from the donor organism, it is placed in yeast, which serves as
the medium for the modifications. Taking advantage of yeast
biology, they were able to modify the genome in ways that could not be
completed in the original host (Mycoplasma mycoides). Ultimately the genome is placed back into a bacterial species (Mycoplasma capricolum) where it forms a new strain with a modified M. mycoides genome. Overall, this study provides a new method for producing modifications that are not possible within the host.
A More In Depth Summary (The Data)
Figure 1. Generation of Type III restriction enzyme deletions (1).
Panel A – The purpose of this panel is to summarize the modifications to the M. mycoides genome they produce. The original genome (YCpMmyc1.1, i) was made into a yeast vector in M. mycoides. The genome was then transformed into yeast (Saccharomyces cerevisiae) along with the knockout cassette. This cassette replaces the typeIIIres
gene through homologous recombination, providing the yeast with a
selectable Ura3 marker as well as a Gal1 controlled expression of the
I-Sce I protein (YCpMmyc1.1-ΔtypeIII::URA3,
ii). Upon expression of this endonuclease, a double stranded cut
occurs a the asterisk and through homologous recombination deletes out
the cassette (red lines represent homologous segments). The
product is a “modification [that] cannot be made with the genetic tools
available for this bacterium” (YCpMmyc1.1-ΔtypeIIIres, iii) .
Panel B – This
panel presents an agarose gel electrophoresis of PCR products from the
three genomes (i-iii) described in Panel A. The primers they used
(P299 and P302) are located just upstream and downstream of the typeIIIres
gene. The first lane (from the left) is a bp ladder. Lanes
2-4 are from PCR reactions on DNA isolated from yeast cells.
Lanes 5-7 are the same DNA constructs as 2-4, except isolated from the
recipient organism (M. capricolum).
The results demonstrate that in both yeast prior to transplanting and
in the bacteria after transplanting, the PCR products appear at
approximately the correct molecular weights on an agarose gel and are
stable within the organism.
Table 1. Transplantation of M. mycoides YCp genomes from yeast into wild-type and RE(-) M. capricolum recipient cells (1).
Upon transplanting the genomes into the recipient cells (wild type M. capricolum),
it was found that the cells did not survive. The authors
hypothesized that this was due to endonuclease activity within the
recipient cells. To overcome this they tried two approaches:
methylating the genome prior to transplanting it or knocking out the
restriction enzyme in the recipient cell.
Both techniques enabled colonies to form. The
authors attempted many combinations of proteins and methylating agents
as well as controls to show that the it was in fact the methylation
that was causing the genome to be transplanted. No colonies
formed in either the untreated samples in wild type hosts or
mock-methylated samples in wild type hosts. It was seen that in
all the restriction enzyme knockouts colonies would form. Their
approach and conclusions are very well presented and defended in the
Figure 2. Southern blot analysis of M. mycoides transplants (1).
Panel A – Does
the DNA change between its native host and the recipient cells?
This southern blot aims to show that the genome is conserved before and
after the modifications to it. The probe in the blot was the
IS1296 sequence, a sequence specific to M. mycoides. The M. capricolum genome
served as a negative control, as it does not contain the IS1296
sequence. Lane 2 shows the typical southern blot with this probe
for the native M. mycoides genome.
The next lanes show the genome after it has been in yeast, either
unmodified or with the different modifications. In all of these
lanes the pattern stays constant, implying that the genome was
conserved throughout their process (minus the modifications that were
demonstrated in Figure 1).
Panel B – Was the typeIIIres gene deleted? The probe in this southern blot is specific to the typeIIIres gene. Wild type M. capricolum
does not contain the gene (as expected), whereas the unmodified native
YCpMmyc1.1 genome does contain it (again as expected). When it
was expected that the deletion would occur (after modification and
transplantation into the recipient), there is no band in the lanes
implying that the gene is in fact gone.
Panel C – To
confirm the deletion occurred correctly, the researchers sequenced the
genome. This panel shows the sequence results from this and shows
that the deletion did occur correctly. Although it is not shown,
they did sequence the entire genome to ensure not only that the
deletion was correct, but also that there were no fragments of the
yeast genome or the recipient cell.
Figure 3. Moving a bacterial genome
into yeast, engineering it, and installing it back into a bacterium by
genome transplantation (1).
This figure presents the overall scheme they used in
their method. In bacteria, DNA is inserted into the chromosome to
make it a yeast vector. This vector is then isolated and placed
in yeast where it can undergo many modifications. Ultimately it
can be placed back into a recipient cell and resolved to create the
new, engineered bacterial strain! Further, as it is a cycle, this
process can be done multiple times to create any number of deletions.
logic of the paper is very coherent, the set out with a goal and have
shown adequate support for the reaching of that goal. That being
said there are a few details left out that would strengthen their
Although Figure 1B is convincing that the changes in
the genome were performed, it lacks a couple controls. None of
the lanes present a negative control, and therefore don’t demonstrate
that the PCR is specific. There are two possible controls, both
of which I believe would be helpful. First, a negative control
with no genomic DNA and just the primers could be run under the same
conditions. Second, run a negative control with genomic DNA from
another species (like mouse) when the primers should not produce a PCR
product from the PCR reaction (if they are specific). This would
demonstrate that the products visualized would are in fact not remnants
of some other reaction.
Did methylation cause the problems transplanting the genome into M. capricolum?
Although their data is logical and convincing, there are another couple
controls that would make their argument more convincing. First,
they state that the deletion of 500 kb serves as a good control as it
deletes essential genes. Yet they only use the control within one
strain of yeast. Further the strain that they use the control in
was not the strain in which they did the majority of their tests.
Even though the two strains of yeast should be very similar, the
control should still be included. If the strain is different, it
is possible that the transformation into the VL6-48N strain would not
provide zero colonies, and therefore its ability as a control would be
proven incorrect. Ideally there would be no colonies and it would
enhance their argument. Considering they have performed a large
number of transformations, one more condition wouldn’t have been too
difficult to manage.
Another interesting approach to this problem would
be to methylate the DNA, then use a demethylating compound and verify
that when it is further removed, the transplantation no longer
works. One could also analyze the protein via gel electrophoresis
to ensure the methyl group was in fact added. On a gel, you would
expect a slightly higher molecular weight compound after treated with
Besides controls, I believe this experiment would be
much stronger if they transformed the genome back into its host
species. Although it is also useful that it can be transplanted
into a different species and therefore make a new strain, many
potential applications would be better if they could transform them
into the original organism. They claim to have used the M. capricolum
strain because of its replication time being faster. Although
this would be a more convenient way to develop this method, ultimately
being able to put it back in the same organism would allow for
approaches such as gene therapy.
This study does not explain the resolution (where
they eliminate the wild type DNA from the recipient cell). There
is no explanation on how that occurred, and it seems to be assumed to
happen. They do point to experiments that confirmed it was gone,
but they don’t say how it disappeared.
Overall, the paper provides good evidence and
coherent logic in their approach to show that genomes can be modified
in non-native species. It has many promising applications and
shows a great proof of concept. No doubt will we see this method
used many times in the future!
What's Up Next?
There are four major areas of future research on the topic that I propose here:
1. Repeat some of the experiments with addition controls.
There is room for improvement within many of the
figures and tables to contain additional controls. Please refer
to the previous section for the suggested experiments and the expected
2. Modify the genome and place it back in the original organism.
This study used a genome from one species and
transplanted it into a different species. Although this is a
proof of concept, the study would be improved through placing it back
into the original species. In doing so they could verify that the
technique would work for making knockouts, adding genes, and
potentially gene therapy.
To do this, the same method should be used (with the
added controls in the previous point of course). The same results
would be expected: the genome should remain in tact and stable between
during the procedure. The appropriate PCR should provide evidence
that the genome was in fact altered.
3. Verify the gene expression in the new system.
There are many methods that can be used to verify the altered gene expression.
First a western blot can analyze whether or not the
protein they knocked out is in fact no longer present. By
obtaining antibodies to the protein of interest, they could show that
protein extracts from the recipient cells after transplantation and
restoration do not contain the protein. This should be done at
different time points after transplantation. If the recipient
cell contained this protein prior to the transplant, at initial time
points the protein may be seen on a western blot until it has been
sufficiently degraded. Overtime the amount of the protein
expression should decrease and ultimately cease (monitored by band
A second method would be an RNA microarray
analysis. This would allow one to screen for the RNA molecules
that were produced from all the genes in the species. Ideally,
the gene that was deleted (typeIIIres)
would not be expressed. This experiment would further be
interesting as it could monitor other changes that may occur because of
the deletion (it monitors many genes at one time). Perhaps the
deleted gene is crucial in some other pathway and without it other gene
expression could cease. This would be a method to look at the
whole genome and ensure that nothing else in the organism is
altered. The ideal results would show that all the gene
expression would stay constant except for the one gene that was
deleted. Nonetheless, it is a way of identifying if any other
changes did occur.
4. Completely synthetic genome.
In their introduction, they referenced creating a
genome from scratch. It would be very interesting to see this
genome actually introduced into the cell, rather than taking a genome
from another species. This should not be too difficult as they
have the genome in yeast, and therefore transplanting it into a cell
would use the same protocol they have developed in this paper. As
the know the sequence of the genome, they should be able to monitor
that it in fact inserted itself appropriately into the organism in the
same manner as described in the paper. This could provide a new
step for synthetic biology as they will be able to create a species
CC. Creating bacterial strains from genomes that have been cloned and
engineered in yeast. Science (New York, N.Y.) 2009;325(5948):1693-6.
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