Synthetic Genomes for Synthetic Biology

178 | Journal of Molecular Cell Biology (2010), 2, 178–179 doi:10.1093/jmcb/mjq015

Research Highlight Synthetic Genomes for Synthetic Biology

Harris H. Wang*

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA * Correspondence to: Harris H. Wang, Tel: +1-617-955-9575; E-mail: [email protected]

A genome synthesized entirely from scratch has been used to replace the native genome of a living cell, thus creating a new cell. This

achievement marks a new frontier in synthetic biology to design and create genomes for organisms with few genetic tools and for Downloaded from https://academic.oup.com/jmcb/article/2/4/178/866681 by guest on 24 September 2021 applications in areas of energy, health care and the environment.

Biologists revel in the excitement of dis- (UGA encodes tryptophan instead of a frameshift error in an essential replication covery, and engineer in the art of creation. stop codon). The design of the synthetic gene (dnaA), which was corrected. In a fusion of two cultures, synthetic biol- genome (JCVI-syn1.0) was based on the To boot up the synthetic genome, Gibson ogists are dissecting the inner workings sequence of an M. mycoides strain the et al. utilized a genome transplantation of life by attempting to recreate it in the JCVI group previously used for genome technique in which the native genome is laboratory, piece by piece. Since the transplantation (Lartigue et al., 2009) entirely replaced with a new genome in a dawn of civilization, humans have been with four additional non-disruptive syn- living host. Prior work showed that fully builders and engineers, constructing thetic watermark sequences. This particu- intact M. mycoides genomic DNA could be houses from bricks, machines from lar genome was chosen for synthesis transferred into an M. capricolum cell by a metals and now genomes from nucleo- because of its modest genome size and polyethylene glycol (PEG)-based chemical tides. As the blue print of life, the fast doubling time (80 min). To construct transformation method (Lartigue et al., genome encodes all the necessary herita- the designed genome, a commercial gene 2007). However, because the synthetic ble information to allow a cell to survive synthesis vendor first generated a M. mycoides genome that propagated as and replicate. Genomes of all living organ- sequence-verified library of 1.1 kb DNA a yeast centromeric plasmid was unmethy- isms replicate based on a preexisting copy. fragments from chemically synthesized oli- lated, it was degraded quickly by the By contrast, de novo synthesis is a new gonucleotides using a strategy first restriction endonuclease system of the paradigm and a powerful approach to described in the 1970s(Khorana et al., recipient M. capricolum cell upon genome create genomes of any sequence, architec- 1972). Then in three hierarchical stages, transplantation. To overcome this issue, ture or design (Figure 1). Gibson et al. assembled the library of in vitro methylation of the synthetic Now for the first time, a genome made 1 kb fragments, which contained 80 bp genome using an M. capricolum cell entirely from chemically synthesized homologous overlaps, first into 10 kb frag- extract or transplantation into a restriction pieces has been successfully booted up ments and then into 100 kb fragments endonuclease-deficient M. capricolum in a living cell at the J. Craig Venter mostly using in vivo homologous recombi- strain proved to be two viable solutions. Institute in a culminating effort that has nation in yeast. Finally, the 11-second- Gibson et al. adopted the latter approach stretched over the past decade. In a techni- stage fragments were assembled into the to isolate tetracycline-resistant blue- cal tour de force, Gibson et al. synthesized whole M. mycoides genome, which propa- colored clones that were putatively former and assembled a 1.08-Mb Mycoplasma gated as a yeast centromeric plasmid in a M. capricolum cells that now contained a mycoides genome de novo and success- yeast clone. DNA sequencing, multiplex transplanted M. mycoides genome. fully transplanted it into a Mycoplasma PCR reactions and restriction digests While the exact mechanism of how the capricolum recipient to create a new were used to sequence-verify the synthetic M. mycoides genome and the M. mycoides cell (Gibson et al., 2010). assembled fragments at each step. native M. capricolum genome resolve in This effort highlights a new breed of syn- To address the bottleneck in rapid the recipient cell remains unknown, PCR thetic biology based on de novo synthesis screening of non-functional assemblies, genotyping, restriction pattern analysis and engineering for creating synthetic Gibson et al. made semi-synthetic and DNA sequencing of the genome from genomes (Carr and Church, 2009). genomes to test functionality of the the self-replicating transplanted cell Mycoplasmas are small commensal or 100 kb intermediates. Through yeast hom- suggested the presence of the designed parasitic bacteria that can cause human ologous recombination of the native M. mycoides genome. Eight unintended respiratory and inflammatory diseases. genome with pieces of the intermediate single nucleotide polymorphisms and These microorganisms lack a cell wall and constructs, the method identified a non- two mutations disrupting nonessential are unique for their altered genetic code viable 100 kb assembly caused by a genes were found through whole-genome

oligonucleotides can be .40%, which can in silico predictions (Feist et al., 2009)of increase to .95% when assembled into physiology, metabolism and regulation 1 kb gene fragments. Gibson et al. found of a synthetic cell will play a crucial role an error frequency of 90% for some assem- for creating useful synthetic genomes. blies of 10 kb fragments. For assemblies of These genomes may contain entirely new 100 kb, the error frequency was 75% with properties (e.g. reassigned genetic small deletions dominating the in vitro codes, rewired regulation, reorganized (e.g. ligation or ampliﬁcation based) or operons) with new phenotypic traits (e.g. in vivo (e.g. yeast homologous recombina- resistance to viruses, modular genome tion) methods. For the 1 Mb assemblies, structure). the success rate was only 2%. Thus, As the synthetic biologist’s toolbox con- errors during genome synthesis can poten- tinues to grow, a new budding branch on

tially accumulate to yield only one error- the Tree of Life is taking shape, emerging Downloaded from https://academic.oup.com/jmcb/article/2/4/178/866681 by guest on 24 September 2021 free genome in 105 or more assembly from new organisms designed, syn- reactions. A rigorous DNA sequencing thesized and created by engineers. These step after each stage of assembly endeavors will require the careful develop- enables efﬁcient removal of error- ment of ethical frameworks around the Figure 1 Genome synthesis requires the containing products, however, at the construction of synthetic life and its poten- assembly and propagation of synthetic DNA fragments, accurate sequence veriﬁcation expense of time and additional resources. tial risk, utility and impact on society. and error correction methods, and the Finally, upon producing a genome free of ability to jumpstart the synthetic genome in synthesis errors, experimental failures in a living cell through transplantation. booting up the genome can arise through a variety of causes such as transplantation References 85 failure or donor/recipient incompatibility. sequencing, including an bp dupli- Carlson, R. (2009). The changing economics of DNA cation and an E. coli IS1 transposon Error detection and correction pipelines synthesis. Nat. Biotechnol. 27, 1091–1094. element (likely from 10 kb fragment are thus crucial for any large-scale Carr, P.A., and Church, G.M. (2009). Genome engin- cloning in E. coli). Morphological studies genome synthesis endeavors and require eering. Nat. Biotechnol. 27, 1151–1162. by electron microscopy and proteomic further improvement. Feist, A.M., Herrgard, M.J., Thiele, I., Reed, J.L., and Palsson, B.Ø. (2009). Reconstruction of bio- 2 While many important technical limit- analysis by D gel-electrophoresis of the chemical networks in microorganisms. Nat. Rev. transplanted cell provided further evi- ations for constructing genomes de novo Microbiol. 7, 129–143. dence that the strain was an M. mycoides. have been resolved by Gibson et al., bar- Forster, A.C., and Church, G.M. (2006). Towards Based on these results, the group con- riers to successfully design and create synthesis of a minimal cell. Mol. Syst. Biol. 2, 45. cluded that the transplanted cell contained new and functional genomes from Gibson, D.G., Glass, J.I., Lartigue, C., Noskov, V.N., Chuang, R.-Y., Algire, M.A., Benders, G.A., the synthetic JCVI-syn1.0 genome. scratch still remain quite high. Even Montague, M.G., Ma, L., Moodie, M.M., et al. The study highlights the important con- though the cost of gene synthesis is drop- (2010). Creation of a bacterial cell controlled by siderations for genome synthesis. As with ping precipitously (Carlson, 2009), the a chemically synthesized genome. Science. any construction project, errors must be present day construction cost is still too 329, 52–56. minimized and readily addressed when high for genome synthesis to be practical. Khorana, H.G., Agarwal, K.L., Bu¨chi, H., Caruthers, M.H., Gupta, N.K., Kleppe, K., Kumar, A., To be able to design a whole genome de encountered. Modes of failure for a syn- Otsuka, E., RajBhandary, U.L., Van de Sande, thetic genome fall mainly into three cat- novo, we need to have deeper under- J.H., et al. (1972). Studies on polynucleotides. egories: in design, in synthesis or in standing of how life’s essential com- CIII. Total synthesis of the structural gene for boot-up. First, poorly designed genomes ponents function and how they interact an alanine transfer ribonucleic acid from yeast. 72 209 217 will have a high likelihood of failure in with one another. Progress towards creat- J. Mol. Biol. , – . Lartigue, C., Glass, J.I., Alperovich, N., Pieper, R., many ways, such as presence of toxic ing a minimal genome may shed light on Parmar, P.P., Hutchison, C.A., III, Smith, H.O., genes, absence of essential genes, or the design principles for creating simpler and Venter, J.C. (2007). Genome transplantation improper genetic regulation. Currently, self-replicating life forms (Forster and in bacteria: changing one species to another. design errors are the most troubling Church, 2006) that can grow in complexity Science 317, 632–638. because risk of failure in new designs is with further engineering over time. For Lartigue, C., Vashee, S., Algire, M.A., Chuang, R.-Y., Benders, G.A., Ma, L., Noskov, V.N., Denisova, still very high as our ability to design a now, we will continue to rely on mixing E.A., Gibson, D.G., Assad-Garcia, N., et al. genome remains in its infant stages. and matching genetic sequences from (2009). Creating bacterial strains from genomes Furthermore, experimental indication of a natural organisms as a way forward that have been cloned and engineered in yeast. bad design only surfaces well after the syn- because our ability to design complex bio- Science 325, 1693–1696. thesis steps. Second, errors can be intro- logical circuits or new proteins de novo is Wang, H.H., Isaacs, F.J., Carr, P.A, Sun, Z.Z., Xu, G., Forest, C.R., and Church, G.M. (2009). still limited. Innovations that enable rapid duced during genome synthesis at one of Programming cells by multiplex genome engin- many steps. The frequency of encountering prototyping of libraries of new genetic eering and accelerated evolution. Nature 460, sequence errors in chemically synthesized designs (Wang et al., 2009) coupled with 894–898.