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SynIII construction. ( A ) BB synthesis. JHU students in the Build-A-Genome course synthesized 750-bp BBs (purple) from oligonucleotides. nt, nucleotides. ( B ) Assembly of minichunks. Two- to 4-kb minichunks (yellow) were assembled by homologous recombination in S. cerevisiae (table S1). Adjacent minichunks were designed to encode overlap of one BB to facilitate downstream assembly steps. Minichunks were flanked by a rare cutting restriction enzyme (RE) site, Xma I or Not I. ( C ) Direct replacement of native yeast chromosome III with pools of synthetic minichunks. Eleven iterative one-step assemblies and replacements of native genomic segments of yeast chromosome III were carried out by using pools of overlapping synthetic DNA minichunks (table S2), encoding alternating genetic markers ( LEU2 or URA3 ), which enabled complete replacement of native III with synIII in yeast.
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Designer Chromosome
One of the ultimate aims of synthetic biology is to build designer organisms from the ground up. Rapid advances in DNA synthesis has allowed the assembly of complete bacterial genomes. Eukaryotic organisms, with their generally much larger and more complex genomes, present an additional challenge to synthetic biologists. Annalur...
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... cerevisiae has a ge- nome size of ~12 Mb distributed among 16 chromosomes. The entire genome encodes ~6000 genes, of which ~5000 are individually nones- sential ( 1 ). Which of these nonessential genes are simultaneously dispensable? Although a number of studies have successfully mapped pairwise “synthet- ic lethal” interactions between gene knockouts, those methods do not scale well to three or more gene combina- tions because the number of combina- tions rises exponentially. Our approach to address this question is to produce a synthetic yeast genome with all nones- sential genes flanked by loxPsym sites to enable inducible evolution and ge- nome reduction (a process we refer to as SCRaMbLEing) in vivo ( 2 , 3 ). The availability of a fully synthetic S. cere- visiae genome will allow direct testing of evolutionary questions—such as the maximum number of nonessential genes that can be deleted without a catastrophic loss of fitness and the catalog of viable 3-gene, 4-gene, ... n - gene deletions that survive under a given growth condition—that are not otherwise easily approachable in a systematic unbiased fashion. Engineer- ing and synthesis of viral and bacterial genomes have been reported in the literature ( 4 – 11 ). An international group of scientists has embarked on constructing a designer eukaryotic genome, Sc2.0 (www.syntheticyeast.org), and here we report the total synthesis of a complete designer yeast chromosome. Yeast chromosome III, the third smallest in S. cerevisiae [316,617 base pairs (bp)], contains the MAT locus determining mating type and was the first chromosome sequenced ( 12 ) . We designed synIII according to fitness, genome stability, and genetic flexibility principles developed for the Sc2.0 genome ( 2 ). The native sequence was edited in silico by using a series of deletion, insertion, and base substitu- tion changes to produce the desired “designer” sequence (Fig. 1, figs. S1 and S2, and supplementary text). The hierarchical wet-laboratory workflow used to construct synIII (Fig. 2) con- sisted of three major steps: (i) The 750- bp building blocks (BBs) were pro- duced starting from overlapping 60- to 79-mer oligonucleotides and assembled by using standard polymerase chain reaction (PCR) methods ( 13 , 14 ) by undergraduate students in the ...
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Abstract
Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native Saccharomyces cerevisiae chromosome III. Changes to...
Citations
... Metabolic engineering often requires the insertion of groups of genes to be expressed with specific individual levels to provide maximum efficiency of the required set of enzymatic reactions while avoiding unnecessary energy cost for production of excessive amounts of proteins [17]. Another trending issue is genome rewriting [18][19][20], which requires changes in synonymous codon sets of protein-encoding genes and may lead to imbalanced production of proteins that harms cell metabolism. ...
An important role of a particular synonymous codon composition of a gene in its expression level is well known. There are a number of algorithms optimizing codon usage of recombinant genes to maximize their expression in host cells. Nevertheless, the underlying mechanism remains unsolved and is of significant relevance. In the realm of modern biotechnology, directing protein production to a specific level is crucial for metabolic engineering, genome rewriting and a growing number of other applications. In this study, we propose two new simple statistical and empirical methods for predicting the protein expression level from the nucleotide sequence of the corresponding gene: Codon Expression Index Score (CEIS) and Codon Productivity Score (CPS). Both of these methods are based on the influence of each individual codon in the gene on the overall expression level of the encoded protein and the frequencies of isoacceptors in the species. Our predictions achieve a correlation level of up to r = 0.7 with experimentally measured quantitative proteome data of Escherichia coli, which is superior to any previously proposed methods. Our work helps understand how codons determine protein abundances. Based on these methods, it is possible to design proteins optimized for expression in a particular organism.
... In eukaryotes, the Synthetic Yeast 2.0 (Sc2.0) consortium, an international project to develop an artificial yeast genome, succeeded in constructing a highly modified and fully functional synthetic version of the baker's yeast chromosome (Annaluru et al. 2014). This was the first project to design and partially complete a eukaryotic genome from scratch. ...
Synthetic biology, an interdisciplinary field at the intersection of engineering and biology, has garnered considerable attention for its potential applications in plant science. By exploiting engineering principles, synthetic biology enables the redesign and construction of biological systems to manipulate plant traits, metabolic pathways, and responses to environmental stressors. This review explores the evolution and current state of synthetic biology in plants, highlighting key achievements and emerging trends. Synthetic biology offers innovative solutions to longstanding challenges in agriculture and biotechnology for improvement of nutrition and photosynthetic efficiency, useful secondary metabolite production, engineering biosensors, and conferring stress tolerance. Recent advances, such as genome editing technologies, have facilitated precise manipulation of plant genomes, creating new possibilities for crop improvement and sustainable agriculture. Despite its transformative potential, ethical and biosafety considerations underscore the need for responsible deployment of synthetic biology tools in plant research and development. This review provides insights into the burgeoning field of plant synthetic biology, offering a glimpse into its future implications for food security, environmental sustainability, and human health.
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... In eukaryotes, the Synthetic Yeast 2.0 (Sc2.0) consortium, an international project to develop an artificial yeast genome, succeeded in constructing a highly modified and fully functional synthetic version of the baker's yeast chromosome (Annaluru et al. 2014). This was the first project to design and partially complete a eukaryotic genome from scratch. ...
Synthetic biology, an interdisciplinary field at the intersection of engineering and biology, has garnered considerable attention for its potential applications in plant science. By exploiting engineering principles, synthetic biology enables the redesign and construction of biological systems to manipulate plant traits, metabolic pathways, and responses to environmental stressors. This review explores the evolution and current state of synthetic biology in plants, highlighting key achievements and emerging trends. Synthetic biology offers innovative solutions to longstanding challenges in agriculture and biotechnology for improvement of nutrition and photosynthetic efficiency, useful secondary metabolite production, engineering biosensors, and conferring stress tolerance. Recent advances, such as genome editing technologies, have facilitated precise manipulation of plant genomes, creating new possibilities for crop improvement and sustainable agriculture. Despite its transformative potential, ethical and biosafety considerations underscore the need for responsible deployment of synthetic biology tools in plant research and development. This review provides insights into the burgeoning field of plant synthetic biology, offering a glimpse into its future implications for food security, environmental sustainability, and human health.
... The question of what actually makes a chromosome a chromosome has fascinated researchers for decades but is now asked from a new perspective with synthetic biology becoming a scientific discipline. It is now possible to construct entire bacterial or eukaryotic chromosomes from synthetic DNA oligonucleotides [1][2][3][4]. This technical revolution raises the question of what engineering rules one has to follow when constructing a chromosome from scratch. ...
... The same fragments with homology can alternatively be transformed into the yeast Saccharomyces cerevisiae to make use of its powerful recombination system to join the respective DNA parts [13]. Assembly in yeast is popular in synthetic chromosome construction, whether bacterial chromosomes or actually synthetic yeast chromosomes [2,[14][15][16]. Another DNA assembly approach making use of short sequence homologies is the ligase cycling reaction (LCR). ...
The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.
... Metabolic engineering often requires insertion of groups of genes to be expressed with specific individual levels to provide maximal efficiency of the required set of enzymatic reactions while avoiding unnecessary energy cost for production of excessive amounts of proteins (17). Another trending issue is genome rewriting (18)(19)(20), which requires changes in synonymous codon sets of protein-encoding genes and may lead to imbalanced production of proteins that could harm cell metabolism. ...
An important role of a particular synonymous codon composition of a gene on its expression level is well-known. There are a number of algorithms optimising codon usage of recombinant genes to maximise their expression in host cells. Nevertheless, the problem has not been solved yet and remains relevant. In the realm of modern biotechnology, directing protein production to a specific level is crucial for metabolic engineering, genome rewriting, and a growing number of other applications. In this study, we propose two new simple statistical and empirical methods for predicting the protein expression level from the nucleotide sequence of the corresponding gene: Codon Expression Index Score (CEIS) and Codon Productivity Score (CPS). Both of these methods are based on the influence of each individual codon in the gene on the overall expression level of the encoded protein and the frequencies of isoacceptors in the species. Our predictions achieve a correlation with experimentally measured quantitative proteome data of Escherichia coli up to a level of r=0.7, which is superior to any previously proposed methods. Our work helps to understand how codons determine translation rates; based on our methods, it is possible to design proteins optimised for expression in a particular organism.
... Mixing polymer families with different reactive ends further enhances the designability, as it introduces different c * for each family. Our results can be used to optimize the conditions for DNA engineering, e.g., transfection vectors [2] ought to be ligated at c/c * < 0.1, whereas synthetic chromosome assemblies [57] at large c/c * . Finally, it may be possible to couple dissipative DNA breakage reactions [48,58,59] with ATP-consuming ligation to create dense solutions of self-sustained topologically active viscoelastic fluids, which would be an interesting active fluid to investigate in the future. ...
The process of polymer condensation, i.e., the formation of bonds between reactive end groups, is ubiquitous in both industry and biology. Here we study generic systems undergoing polymer condensation in competition with cyclization. Using a generalized Smoluchowski theory, molecular dynamics simulations and experiments with DNA and ATP-consuming T4 ligase, we find that this system displays a transition, from a ring-dominated regime with finite-length chains at infinite time to a linear-polymers-dominated one with chains that keep growing in time. Finally, we show that fluids prepared close to the transition may have widely different compositions and rheology at large condensation times.
Published by the American Physical Society 2024
... While the lower complexity of the system might be viewed as a limitation, it can also serve as an advantage for isolating and analyzing specific components of complex entities. Conversely, the yeast system is the only eucaryote for which synthetic biology is currently possible at the genome-wide scale (Annaluru et al., 2014). ...
... To facilitate characterization of the relationship between genotypes and phenotypes and confer synthetic chromosome the ability to evolve, we introduced two genetic manipulation systems in the synAC: an orthogonal random rearrangement system and a precise gene editing system. Learning from the Sc 2.0 project, 32,33 for the design of the orthogonal rearrangement system, we introduced numerous orthogonal rearrangement sites (loxP and vox) into the synAC. The vox sites were placed upstream of each non-homologous gene and the loxP sites were placed at equivalent positions in the region of orthologous genes. ...
Synthetic biology confers new functions to hosts by introducing exogenous genetic elements, yet rebuilding complex traits that are based on large-scale genetic information remains challenging. Here, we developed a CRISPR/Cas9-mediated haploidization method that bypasses the natural process of meiosis. Based on the programmed haploidization in yeast, we further developed an easy-to-use method designated HAnDy (Haploidization-based DNA Assembly and Delivery in yeast) that enables efficient assembly and delivery of large DNA, with no need for any fussy in vitro manipulations. Using HAnDy, a de novo designed 1.024 Mb synthetic accessory chromosome (synAC) encoding 542 exogenous genes was parallelly assembled and then directly transferred to six phylogenetically diverse yeasts. The synAC significantly promotes hosts’ adaptations and increases the scope of the metabolic network, which allows the emergence of valuable compounds. Our approach should facilitate the assembly and delivery of large-scale DNA for expanding and deciphering complex biological functions.
... To verify if repetitive usage of the GEM construct caused genomic instability, we used ySTART (biological quadruplicates) in a short-term evolution experiment, similar to previous methods 101 . We grew ON cultures in 5 mL YPD in test tubes and inoculated at OD 600 nm 0.05 at day 0. Strains were grown for 15 days and diluted (1/1000) to fresh medium at different time points (day 1, day 2, day 3, day 5, day 6, day 7, day 9, day 12, day 13). ...
Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.
... Subsequently, in 2010, the Venter Institute synthesized the first self-replicating artificial cell, Mycoplasma capricolum [3]. More recently, in 2017, the Synthetic Yeast Genome Project (Sc2.0), an international collaboration, achieved the de novo design and chemical synthesis of six chromosomes (II [4], III [5], V [6], VI [7], X [8] and XII [9]) of the S. cerevisiae genome. The synthetic yeast strains were remarkably similar and consistent with the wild type. ...
DNA synthesis and assembly primarily revolve around the innovation and refinement of tools that facilitate the creation of specific genes and the manipulation of entire genomes. This multifaceted process encompasses two fundamental steps: the synthesis of lengthy oligonucleotides and the seamless assembly of numerous DNA fragments. With the advent of automated pipetting workstations and integrated experimental equipment, a substantial portion of repetitive tasks in the field of synthetic biology can now be efficiently accomplished through integrated liquid handling workstations. This not only reduces the need for manual labor but also enhances overall efficiency. This review explores the ongoing advancements in the oligonucleotide synthesis platform, automated DNA assembly techniques, and biofoundries. The development of accurate and high-throughput DNA synthesis and assembly technologies presents both challenges and opportunities.