*This web page was produced as an assignment for an undergraduate course at Davidson College.*
Davidson College Molecular Biology
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, Hamilton O. Smith, J. Craig Venter, John I. Glass
As published in Science, Vol. 325: 1693-1696. 25 September 2009.
Overview
Lartigue et al. describe their success in isolating the genome of one bacterial strain, transplanting it into yeast for genome modification, and inserting the altered genome into a completely different bacterial strain. The lab isolated the genome of Mycoplasma mycoides, altered it, inserted it into the yeast Saccharomyces cerevisiae, further altered the genome, and inserted the altered genome into Mycoplasma capricolum, thereby transforming it into M. mycoides. Their paper highlights the methods necessary to carry a genome between prokaryotes and eukaryotes and illustrates the use of yeast to carry out genome modifications not possible in bacteria. At its core the paper provides a powerful new tool for molecular and synthetic biologists.
In-Depth Summary
Figure 1A: This genomic flow chart shows the modifications done to the M. mycoides genome in yeast. Such genetic modifications are not possible in M. mycoides. YCpMmyc1.1 is the name of the transformed M. mycoides genome inserted into yeast. M. mycoides was transformed with a vector encoding for tetracycline resistance, b-galactosidase, and a yeast auxotrophic marker, centromere and autonomously replicating sequence. Figure 1A(i) shows a 3.1 kb segment of this genome containing a Type III restriction enzyme gene (typeIIIres) that was targeted for deletion to create Figure 1A(iii). First a knockout cassette containing the Gal1 promoter, SCE1 endonuclease gene, the URA3 gene, and a tandem repeat sequence was used to transform the yeast containing YCpMmyc1.1. Growth on a (His)-, (Ura)- medium selected for yeast containing Figure 1A (ii). Growth on galactose promotes the production of SCE1 endonuclease which cuts at the asterisk shown and allows for the recombination of the tandem repeats noted by the red bars. Further growth on a 5-FOA medium selects for the final deletion product YCpMmyc1.1-ΔtypeIIIres. URA3 is essential for the synthesis of pyrimidines, but can also convert the non-toxic 5-FOA into its toxic form 5-fluorouracil. Thus any yeast that did not undergo the deletion would die.
Figure 1B: Lartigue et al. claim that this gel electrophoresis validates the genomic modifications of Figure 1:A. Using the two primers, P299 and P302, indicated in Figure 1A, they performed PCR to obtain bands corresponding to YCpMmyc1.1 (i), YCpMmyc1.1-ΔtypeIIIres::URA3 (ii), and YCpMmyc1.1-ΔtypeIIIres (iii). Lartigue et al. performed PCR on DNA from both yeast and from the post-transplant (the M. capricolum turned M. mycoides). The gel shows that for both yeast and the post-transplant, the PCR products of i, ii, and iii have the expected molecular weights relative to one another. Lane i has a molecular weight around 3 kb, lane ii, with the deletion of typeIIIres and the insertion of the cassette, has a slightly larger molecular weight, and lane iii, with the complete deletion, has the smallest molecular weight by far. Further the bands line up in both the yeast and the post-transplant and thereby support the conclusion that the modified genome had been transferred.
Table 1: Lartigue et al. codify the results of transplanting the YCpMmyc1.1 genome from yeast into M. capricolum. When YCpMmyc1.1 was inserted into the wild type M. capricolum cells, no colonies grew. Lartigue et al. hypothesized that this was due to the presence of restriction endonucleases (which were digesting the transplanted DNA) in the M. capricolum cells. To fix this, two methods were applied. First an M. capricolum straing was developed without restriction endonuclease (RE) activity. Second, the transplanted DNA was protected via in vitro methylation. Using the yeast strain VL6-48N, the YCpMmyc1.1 genome was methylated either from M. capricolum extracts, M. mycoides extracts, M. mycoides purified methylases, or through mock methylation. Both the methlation and the removal of RE activity allowed for transplanted M. capricolum growth. Without RE activity all transplants were able to grow in similar amounts except for the YCpMmyc1.1-Δ 500kb transplant. This genome lacked 500 kb of essential M. mycoides genes and thus served as the negative control. Methylation was also enough to protect the transplanted genome except for the mock methylated genomes. The mock methylated YCpMmyc1.1 genome produced zero colonies. The table also shows that the YCpMmyc1.1 genome modified in yeast were able to produce M. capricolum transplant colonies, at least in cells without RE activity. Methylation was not attempted on the genome altered in yeast.
Figure 2A: This Southern blot analysis uses a M. mycoides specific probe. The blot probed wild type M. capricolum (non-transplanted), M. mycoides YCpMmyc1.1 (non-transplanted) and the transplants YCpMmyc1.1, YCpMmyc1.1-ΔtypeIIIres::URA3, and YCpMmyc1.1-ΔtypeIIIres. As expected, the wild type M. capricolum did not have a band, but any organism with a M. mycoides genome did. Lartigue et al. produced this blot to show their transplants are in fact M. mycoides.
Figure 2B: Lartigue et al. use this Southern blot analysis to argue that the genetic alteration, the deletion of the typeIIIres gene, in yeast did occur and that it already does not exist in wild type M. capricolum. Using the typeIIIres gene as a probe, only the native M. mycoides YCpMmyc1.1 genome has a band. Both the YCpMmyc1.1-ΔtypeIIIres::URA3, and YCpMmyc1.1-ΔtypeIIIres genomes have the typeIIIres gene deleted and thus should not have a band, which the blot confirms.
Figure 2C: In order to show that the transplants genome was completely M. mycoides, Lartigue et al. sequenced the genome of a YCpMmyc1.1-ΔtypeIIIres transplant. The sequence shown is the deletion region of that transplant. Most notable is that the typeIIIres gene has been deleted except for a 60 bp sequence (in blue) which Lartigue et al. contribute to the overlap between the type111res gene and the preceding typeIIImod gene (see Figure 1A:i).
Figure 3: Lartigue et al. summarize the transplant procedure with this diagram. The yeast vector is added to a bacterial genome, which is then placed in yeast, and genetically modified. That modified genome is methylated and placed in a recipient cell. Resolution of that transplanted cell results in a completely engineered bacterium.
Critique (Keep in mind that as an undergraduate, my knowledge and experience are limited and I still respect the excellence of this work)
1) Figure 1B lacks a negative control. There is no evidence that the primers used are specific to the YCpMmyc1.1 genome. Ideally a lane should have been run with genomic M. capricolum DNA.
2) Throughout the paper there are claims of colony growth, yet no evidence is presented beyond the numbers in Table 1. It would have been nice to have images of colony growth (of course these might have been in the supplement) or proof of colony growth. Perhaps a Northern blot or Western blot for a gene/protein specific to M. mycoides could be used to show that the transplanted cell is completely functioning i.e. that transplanting did not interfere with the genes of M. mycoides.
3) While Table 1 displays the negative control, the 500 kb deletion, for the W303a strain, there is no negative control for the VL6-48N strain. Further why was methlyation not attempted on the genomes altered in yeast to see if they too could grow in wild-type M. capricolum? Does methylation still work when the genomic modification has occurred using yeast tools?
4) While in the text Lartigue et al. state that the transplant M. mycoides genome was identical to the original M. mycoides genome there is no evidence of this. Instead of a sequence comparison the authors present a region of DNA and state it is what they say it is.
5) The biggest question unanswered by Lartigue et al. concerns resolution. Resolution would seem to be a crucial step. If all of the M. capricolum genome is not removed it could interfere with the transplanted genome. The authors present no evidence as to what happens to the M. capricolum genome. Figure 3 seems to suggest that it is somehow removed from the recipient cell, but no information is presented as to how. They stated that their sequencing proved that it was not in the cell, so where is it and how did it get there? How were they able to guarantee that none of the M. capricolum genome remains?
Further Research
Gibson et al. have reported the complete chemical synthesis of a bacterial genome and its assembly in yeast. The method outlined in Lartigue et al. provides the means to isolate this synthetic genome and introduce it into a bacterium, to create a fully functional organism. The next step would be to actually carry this out. The procedure outlined in Lartigue et al. could be followed except that instead of the genome originating from M. mycoides it would originate from the synthetic. Also how much modification can be done in yeast and still produce a functioning transplant? If the essential genes are left intact, how many new genes can be added? For this work, Lartigue et al. use only the genus, Mycoplasma for both the transplant DNA and the recipient cell. Will this method work outside of Mycoplasma? Does both the transplant DNA and recipient cell have to be from the same genus or could the M. mycoides genome be transplanted into E. coli? In essence, what are the limits of the method? Finally it will be interesting to see whether this could be a new way for drug synthesis or biofuel production.
References
1) Lartigue, C, Vashee, S, Algire, MA, Chuang, RY, Benders, GA, Ma, L, Noskov, VN, Denisova, EA, Gibson, DG, Assad-Garcia, N, Alperovich, N, Thomas, DW, Merryman, C, Hutchison, CA 3rd, Smith, HO, Venter, JC, Glass, JI. 2009 September. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science; 325 (5948): 1693-1696. PubMed
2) Gibson, DG, Benders, GA, Andrews-Pfannkoch, C, Denisova, EA, Baden-Tillson, H, Zaveri, J, Stockwell, TB, Brownley, A, Thomas, DW, Algire, MA, Merryman, C, Young, L, Noskov, VN, Glass, JI, Venter, JC, Hutchison, CA 3rd, Smith, HO. 2008 February. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science; 319 (5867): 1215-1220. PubMed
Davidson College Molecular Biology
Questions? Comments? E-mail Bobby DesPain.