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A Review of:

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

Carole 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 data.

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.

  A Critique...

    The 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 argument. 
    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 the methylases.
    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 outcomes.

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 intensity).
    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 from scratch.


(1)  Lartigue 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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