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Paper Review: Synthesis of a Designer Chromosome
Research Question and Approach: Annaluru et al.(2014) sought to synthesize a complete functional chromosome based on chromosome III in the eukaryotic organism Saccharomyces cerevisae (yeast). By synthesizing a complete, functional chromosome, researchers will be able to directly determine which nonessential genes can be deleted simultaneously with minimal effect on fitness in a given growth condition. Current methods only allow feasible analysis of the effects of 2 or fewer gene deletions, and only viral and bacterial genomes have been synthesized thus far. The researchers' designer chromosome, synIII, is based on chromosome III in yeast and was selected due to its small size and its mating type determination locus MAT. SynIII was designed to differ from the native chromosome in several ways: its stop-codon replacements, deletions of introns, tRNAs, transposons, and silent mating loci as well as the insertion of loxPsym sites for genome SCRaMbLEing ("synthetic chromosome rearrangement and modification by loxP-mediated evolution") (Dymond et al., 2011). To build the chromosome, overlapping building blocks were assembled into larger overlapping minichunks which were then used to replace native chromosome III DNA via homologous recombination during transformations. Once the chromosome had been designed, the researchers used PCRtag, karyotypic, and phenotypic analyses to characterize the yeast strain with the synIII chromosome. Finally, they assessed genome integrity through loss rate analysis and genome stability through scrambling via the loxPsym sites.
Conclusions: After confirming the successful replacement of native chromosome III with synthetic chromosome III through PCRtag analysis, researchers determined that the synIII chromosome was functional in yeast and not associated with any negative fitness effects when used instead of the native chromosome III in a yeast strain. Despite the many changes made to synIII, synIII strains were demonstrated to be highly stable, and the synIII chromosome was not found to be lost from yeast cells at a significantly higher rate than the native chromosome III was. Furthermore, the researchers tested their design to demonstrate the effects of scrambling the synIII chromosome on the loss of the MAT locus via estradiol. These researchers carefully designed a fully functional eukaryotic chromosome that could be used to test the effects of nonessential gene deletion. The method used to design this chromosome may also be applicable in establishing a fully synthetic genome.
This paper is really quite interesting. It does an excellent job explaining the method of design, and, from the perspective of the broader scientific community, that is a very important part of this paper. Annaluru et al. clearly tested their design in a variety of different capacities, and they seemed to do a good job in selecting which data to use to demonstrate their success. Figures are clearly depicted and captioned, and the significance of study is clearly conveyed in the body of the paper.
At the same time, it feels as though the limited space could be used more efficiently with respect to both research presentation and content. For example, the depiction of the method of generation development in Figure 4A seems to be very basic. It is likely that the readers would understand the method without needing an illustration. In contrast, the idea of the MATα/a locus in described in completely inadequate detail. The significance of that locus and the a-like faker assay should be described or demonstrated more carefully. Similarly the method of SCRaMbLEing should be explained at some point as well, given that it is an important part of function testing and that it relates to the insertion of the loxPsym sites during chromosome synthesis. I also feel that the data in Figure 3C should be displayed numerically. Not much can be gained by looking at pictures of plates. Either the figure should be omitted (given the sufficient description in the body of the paper), or colony size should be numerically provided for each condition and strain. The (C) in the Figure 3C caption should also be bolded. Finally, the terminology referring to the intermediate strain as well as the synIII left arm could be made clearer. Collectively, the paper demonstrates important information, but slight changes to the content could improve the power of the article.
Figure 1: This figure is a representative display of the synIII design. The designer chromosome is a compilation of non-essential, uncharacterized, dubious, and essential open reading frames (ORFs) as well as repeats, according to the key (A). Researchers altered the native chromosome III by embedding loxPsym sites along the chromosome (A) and specifically within the untranslated region (B) as indicated by the green diamonds. The TAG codon was also altered to a TAA codon, serving as a synthetic stop codon (C). This visual sample of synIII details the changes made to the sequence of native chromosome to design synIII.
Figure 2: This conceptual figure diagrams the methodology of designer chromosome construction. (A) Short, overlapping nucleotides were were assembled with PCR to form 750-bp building blocks that were then ligated into plasmids and transformed into cells. (B) Through homologous recombination, the overlapping building blocks were assembled into larger 2-4kb minichunks, each ligated into a shuttle vector and flanked by restriction enzyme cutting sites. (C) Again, homologous recombination was used along with 2 genetic markers to replace the native genome with overlapping minichunks. Ultimately, 11 replacements were completed to replace the full native chromosome with the synIII.
Figure 3A: To ensure successful construction of the synIII chromosome, the researchers tested the left arm of synIII (“synIII gDNA”) and wildtype (“WT gDNA”) for the presence of markers (PCRtags, top labels) unique to the synthetic or WT strain. No WT markers were present in the synIII chromosome arm, and no synthetic markers were present in the WT arm. Because the synthetic arm did not contain demonstrate any native PCRtags, the researchers concluded that they had successfully replaced the native chromosome with their designer, synthetic chromosome.
Figure 3B: Pulsed-field gel electrophoresis revealed the karyotypes of the native strain (WT), an intermediate strain of synthetic left arm (left arm*), and the synIII strain. Chromosomes IX, VI, and I were found to be of the same size as they migrated the same distance. However, native chromosome III (III) was found to be larger than intermediate III (synIII left arm*) which was in turn larger than chromosome III in the synIII strain (synIII). Given that synIII’s chromosome III contained many deletions relative to the WT strain’s chromosome, this analysis confirmed the expected size reduction and synthetic design.
Figure 3C: In order to compare fitness of the WT strain, the intermediate synIIIL strain, and the synIII strain of yeast, the researchers analyzed colony size of these strains when grown under different conditions. The phenotypes of the colonies from the 3 strains were indistinguishable when grown on 3 different types of media (YPD, YPGE, and MMS), in 2 different pH treatments (4.0 and 9.0), and in 3 different temperatures (25°C, 30°C, and 37°C). These results indicate that the synIII modifications did not cause any unintended changes in fitness, as assessed by colony size.
Figure 4A: Annaluru et al. found that the synIII strain was stable over time, with a very low level of predicted segment loss frequency per generation, after analyzing segment loss of 30 independent synIII strain lineages. As indicated pictorially, they grew 30 independent lineages of synIII for 10 days, transferring lineages each day to new YPD media, for analysis of segment loss after 125 mitotic generations. PCRtag analysis of 58 non-essential segments after gDNA prep revealed no deletions. With a predicted frequency of segment loss per generation of < 4.6 x 10-6, this synthetic strain has strong genome integrity and is highly stable.
Figure 4B: To further assess the stability of the synIII strain, researchers used an a-like faker assay to compare the loss rates of the synIII strain with the WT strain. WT and synIII strains both with MATα locus on their chromosome III or synIII were plated to a lawn of MATα cells. The ability to mate and produce colonies on the plate is only possible if the MAT locus has been lost, or in this case, the WT chrosome III or synIII has been lost, as MAT-less cells default to MATa-specific mating. Thus, the loss rate of chromosome III for both WT and synIII cells was calculated based on the colonies that were able to mate as MATa on the MATα plate due to a loss of chromosome III. Because no significant difference was identified in the loss of chromosome III between WT and synIII-carrying strains, the synthetic synIII chromosome is not lost at a significantly higher rate than the WT chromosome III.
Figure 4C: Researchers used diploid cells that were heterozygous for the MAT locus (MATα/a) to determine the impact of induced genome scrambling via estradiol. Diploid cells with MATα between loxPsym sites (diamonds) on a synIII chromosome and MATa on the native chromosome III (“synIII/III heterodiploid”) were compared in mating types after genome rearrangement via SCRaMbLE to WT MATα/a diploids (“III/III”). Post-scrambling, the synIII/III strain gained the ability to mate as a MATa cells through loss of the MATα locus, indicated by the strong increase in a-mater frequency for this strain compared to the III/III strain. The SCRaMbLE method thus successfully induces loss of segments of synIII via the loxPsym sites associated with genome rearrangement and, as a result, changes the mating phenotype.
Annaluru, N., et al. 2014. Total synthesis of a functional designer eukaryotic chromosome. Science. 344:55-58.
Dymond, J., et al. 2011. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature. 477:471-476.
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