We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within
(
), ...encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the
transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.
Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including ...phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by
-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.
We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in
SynXII was assembled using a two-step method, specified by successive megachunk ...integration and meiotic recombination-mediated assembly, producing a functional chromosome in
Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued 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 used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a
synXII strain that would be identified as
by standard DNA barcoding procedures.
Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as ...707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in
, and a loxPsym site affecting promoter function of
PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.
•Applying the MCDM process for MUT location selection based on spatial data.•Defining the criteria that influence the MUT location selection.•Defining the required GIS datasets for quantifying the ...score of each street segment.•Comparing the ranking results AHP and ANP combined with TOPSIS.•Developing a prototype system to integrate the MCDM methods in a GIS platform.
Multi-purpose utility tunnels (MUTs) integrate all underground utilities in one accessible tunnel. MUTs reduce the need for excavations and their associated costs, as well as the resulting traffic congestion. Several MUTs have been implemented in different parts of the world. Their locations have either been politically influenced or selected to preserve heritage sites or to meet the conditions of a newly developed city. Nevertheless, selecting the location in an existing city under street segments is affected by several criteria that have different spatial characteristics. Combining these characteristics and managing the trade-offs that exist between them determine the ranking of alternative MUT locations. The use of subjective and objective weights in the decision-making process will offer different perspectives from the decision-maker's perspective and the data itself, respectively. This paper aims to analyze spatial data as an input in the multi-criteria decision-making (MCDM) process of the MUT location selection. The objectives are: (1) defining the criteria that influence the MUT location selection, (2) defining the required GIS datasets for quantifying the criteria as scores for each candidate street segment, (3) analyzing the impacts of the dependencies between the criteria by comparing the ranking results of two MCDM methods (i.e., Analytic Hierarchy Process (AHP) and Analytic Network Process (ANP)) combined with the Technique for Order Preference by the Similarity to Ideal Solution (TOPSIS), (4) analyzing the difference between using subjective weights or objective weights, and (5) developing a prototype system to integrate the MCDM methods in a GIS platform. A vector-based spatial analysis is conducted to identify the suitable locations for MUT construction based on 12 criteria representing physical condition information or affecting social costs. Two subjective MCDM methods (i.e., AHP and ANP) are used to generate each criterion's weights, and the ranking of alternatives is determined using TOPSIS. Another set of weights representing the objective weights are calculated for each criterion using the Shannon Entropy method. These weights are combined with TOPSIS to obtain an objective ranking of the alternatives. Based on the results from the different combinations (AHP + TOPSIS, ANP + TOPSIS, and ENTROPY + TOPSIS), the top alternative is always the same.
Although the design of the synthetic yeast genome Sc2.0 is highly conservative with respect to gene content, the deletion of several classes of repeated sequences and the introduction of thousands of ...designer changes may affect genome organization and potentially alter cellular functions. We report here the Hi-C-determined three-dimensional (3D) conformations of Sc2.0 chromosomes. The absence of repeats leads to a smoother contact pattern and more precisely tractable chromosome conformations, and the large-scale genomic organization is globally unaffected by the presence of synthetic chromosome(s). Two exceptions are synIII, which lacks the silent mating-type cassettes, and synXII, specifically when the ribosomal DNA is moved to another chromosome. We also exploit the contact maps to detect rearrangements induced in SCRaMbLE (synthetic chromosome rearrangement and modification by
-mediated evolution) strains.
It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due ...to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts.
Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair ...chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in
under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.
Computational design tools are the cornerstone of synthetic biology and have underpinned its rapid development over the past two decades. As the field has matured, the scale of biological ...investigation has expanded dramatically, and researchers often must rely on computational tools to operate in the high-throughput investigational space. This is especially apparent in the modular design of DNA expression circuits, where complexity is accumulated rapidly. Alongside our automated pipeline for the high-throughput construction of Extensible Modular Mammalian Assembly (EMMA) expression vectors, we recognized the need for an integrated software solution for EMMA vector design. Here we present EMMA-CAD (https://emma.cailab.org), a powerful web-based computer-aided design tool for the rapid design of bespoke mammalian expression vectors. EMMA-CAD features a variety of functionalities, including a user-friendly design interface, automated connector selection underpinned by rigorous computer optimization algorithms, customization of part libraries, and personalized design spaces. Capable of translating vector assembly designs into human- and machine-readable protocols for vector construction, EMMA-CAD integrates seamlessly into our automated EMMA pipeline, hence completing an end-to-end design to production workflow.
Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of ...Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.
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•Designed, built, and characterized a neochromosome with 275 tRNA genes in yeast•Additional tRNA genes burden the host cell, causing deletions or increased ploidy•Chemical tRNA neochromosome extraction enables transplantation into new yeast strains•Functional analysis reveals insights into tRNA and chromosomal biology
A designer tRNA neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae reveals unexpected genomic plasticity and insights into both tRNA and chromosome biology.
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