We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross." Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

https://science.sciencemag.org/content/sci/355/6329/1040.full.pdf

Design of a synthetic yeast genome

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.

Total synthesis of Escherichia coli with a recoded genome

Recent development in electrocatalysts for hydrogen production through water electrolysis

INTRODUCTION The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis. RATIONALE Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders. RESULTS We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University. We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design. We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV. CONCLUSION The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders.

http://science.sciencemag.org/content/sci/355/6329/eaaf4704.full.pdf

"Perfect" designer chromosome v and behavior of a ring derivative

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.

Creating a functional single-chromosome yeast

INTRODUCTION It has long been an interesting question whether a living cell can be constructed from scratch in the lab, a goal that may not be realized anytime soon. Nonetheless, with advances in DNA synthesis technology, the complete genetic material of an organism can now be synthesized chemically. Hitherto, genomes of several organisms including viruses, phages, and bacteria have been designed and constructed. These synthetic genomes are able to direct all normal biological functions, capable of self-replication and production of offspring. Several years ago, a group of scientists worldwide formed an international consortium to reconstruct the genome of budding yeast, Saccharomyces cerevisiae . RATIONALE The synthetic yeast genome, designated Sc2.0, was designed according to a set of arbitrary rules, including the elimination of transposable elements and incorporation of specific DNA elements to facilitate further genome manipulation. Among the 16 S. cerevisiae chromosomes, chromosome XII is unique as one of the longest yeast chromosomes (~1 million base pairs) and additionally encodes the highly repetitive ribosomal DNA locus, which forms the well-organized nucleolus. We report on the design, construction, and characterization of chromosome XII, the physically largest chromosome in S. cerevisiae. RESULTS A 976,067–base pair linear chromosome, synXII, was designed based on the native chromosome XII sequence of S. cerevisiae , and chemically synthesized. SynXII was assembled using a two-step method involving, successive megachunk integration to produce six semisynthetic strains, followed by meiotic recombination–mediated assembly, yielding a full-length functional chromosome in S. cerevisiae. Minor growth defect “bugs” detected in synXII were caused by deletion of tRNA genes and were corrected by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit. The same synthetic rDNA unit was also used to regenerate rDNA at three distinct chromosomal locations. The rDNA signature sequences of the internal transcribed spacer (ITS), often used to determine species identity by standard DNA barcoding procedures, were swapped to generate a Saccharomyces synXII strain that would be identified as S. bayanus. Remarkably, these substantial DNA changes had no detectable phenotypic consequences under various laboratory conditions. CONCLUSION The rDNA locus of synXII is highly plastic; not only can it be moved to other chromosomal loci, it can also be altered in its ITS region to masquerade as a distinct species as defined by DNA barcoding, used widely in taxonomy. The ability to perform “species morphing” reported here presumably reflects the degree of evolutionary flexibility by which these ITS regions change. However, this barcoding region is clearly not infinitely flexible, as only relatively modest intragenus base changes were tolerated. More severe intergenus differences in ITS sequence did not result in functional rDNAs, probably because of defects in rRNA processing. The ability to design, build, and debug a megabase-sized chromosome, together with the flexibility in rDNA locus position, speaks to the remarkable overall flexibility of the yeast genome.

http://science.sciencemag.org/content/sci/355/6329/eaaf3981.full.pdf

Engineering the ribosomal DNA in a megabase synthetic chromosome

The bacterium Escherichia coli remains the leading host for protein expression in large quantity for the purpose of crystallization or other biochemical studies. However, expression of multicomponent protein complexes remains a challenge, and is often laborious and time-consuming. Here we developed a method named EcoExpress, which allows efficient construction of plasmids to express individual protein with user-defined epitope-tag, followed by one-pot assembly of a single vector to express the entire protein complex for copurification. A versatile set of vectors was designed to provide various choices to control the expression of a protein with different promoters, and to accept different number of components for coexpression. Using EcoExpress, we demonstrated that each subunit within a protein complex could be expressed individually or simultaneously, and the entire complex could be copurified. In addition, to overcome the decreased assembly efficiency with the increasing number of components, a novel ol...

EcoExpress-Highly Efficient Construction and Expression of Multicomponent Protein Complexes in Escherichia coli.

Effects of Nb and Mo on 1200 °C Steam Oxidation and Mechanical Properties of FeCrAl Alloys for Fuel Cladding Materials

Impact of Sub-Grain Structure on Radiation Resistance in Additively Manufactured 316l Stainless Steels: An Atomic Insight into the Mechanism

The invention discloses a saccharomyces cerevisiae chromosome as well as a construction method and application thereof. The saccharomyces cerevisiae chromosome comprises two telomeric repeat sequences, two insulator sequences, a centromere-autonomously replicating sequence, a po130 gene and n repeated recombination boxes. Experiment results prove that auxotrophic marker genes of the saccharomyces cerevisiae chromosome are few, normal and stable growth in the situation that screening is not carried out can be realized, the expression quantity and copy number of exogenous genes in yeast are increased, the stability of exogenous genes in yeast is improved, genes integrated into a genome can be increased or reduced, and even expanded into new polygene clusters, and the rapid exogenous expression of beta carotene and violacein is successfully realized in yeast, so that a rapid feasible method is provided for yeast heterogeneous production of various exogenous approaches, and the saccharomyces cerevisiae chromosome can meet the application requirements of fields of gene engineering, metabolic engineering and synthetic biology.

Saccharomyces cerevisiae chromosome as well as construction method and application thereof

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Engineering the ribosomal DNA in a megabase synthetic chromosome

EcoExpress-Highly Efficient Construction and Expression of Multicomponent Protein Complexes in Escherichia coli.

Effects of Nb and Mo on 1200 °C Steam Oxidation and Mechanical Properties of FeCrAl Alloys for Fuel Cladding Materials

Impact of Sub-Grain Structure on Radiation Resistance in Additively Manufactured 316l Stainless Steels: An Atomic Insight into the Mechanism

Saccharomyces cerevisiae chromosome as well as construction method and application thereof