SynBioFromLeukipposInstitute

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress activated transcription factor, activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly-used food additive and Nrf2 activator, tert-butylhydroquinone (tBHQ), suppresses IL-2 production, CD25 expression, and NFκB activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using CRISPR/Cas9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NFκB activation in a Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NFκB activation in other models. These results lead us to investigate another, more potent, Nrf2 activator, the synthetic triterpenoid CDDO-Imidazolide (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in a Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NFκB and Nrf2.

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.

Structured Abstract
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.

Structured Abstract
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.

A core theme in synthetic biology, “understanding by creating,” inspired the effort to generate the first synthetic cell, JCVI-Syn1.0 (1). The project Sc2.0 is elevating this concept by attempting to create a synthetic version of a more evolved organism, Saccharomyces cerevisiae, a eukaryotic single-celled yeast. In a set of papers in this issue (2–8), scientists of the Sc2.0 project who previously constructed a single yeast chromosome (9) now report constructing five additional yeast chromosomes (more than one-third of the entire genome) (see the photo). Using a variety of phenotypic assays and structural and functional genomics techniques, the researchers observed that the synthetic chromosomes drive biological processes just like the natural, native chromosomes.

The assembly of individual protein subunits into large-scale structures is important in many biological contexts. Proteins may assemble into geometrical cages or extended lattices that are characterized by a high degree of symmetry; examples include viral capsids and bacterial S-layers. The precisely defined higher order structure exhibited by these assemblies has inspired efforts to design such structures de novo by applying the principles of symmetry evident in natural protein assemblies. Here we discuss progress towards this goal and also examples of natural protein cages and lattices that have been engineered to repurpose them towards a diverse range of applications in materials science and nano-medicine.

The use of abstract chemical reaction networks (CRNs) as a modelling and design framework for the implementation of computing and control circuits using enzyme-free, entropy driven DNA strand displacement (DSD) reactions is starting to garner widespread attention in the area of synthetic biology. Previous work in this area has demonstrated the theoretical plausibility of using this approach to design biomolecular feedback control systems based on classical proportional-integral (PI) controllers, which may be constructed from CRNs implementing gain, summation and integrator operators. Here, we propose an alternative design approach that utilises the abstract chemical reactions involved in cellular signalling cycles to implement a biomolecular controller - termed a signalling-cycle (SC) controller. We compare the performance of the PI and SC controllers in closed-loop with a nonlinear second-order chemical process. Our results show that the SC controller outperforms the PI controller in terms of both performance and robustness, and also requires fewer abstract chemical reactions to implement, highlighting its potential usefulness in the construction of biomolecular control circuits.

Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.

Structured Abstract
INTRODUCTION
Total synthesis of designer chromosomes and genomes is a new paradigm for the study of genetics and biological systems. The Sc2.0 project is building a designer yeast genome from scratch to test and extend the limits of our biological knowledge. Here we describe the design, rapid assembly, and characterization of synthetic chromosome VI (synVI). Further, we investigate the phenotypic, transcriptomic, and proteomic consequences associated with consolidation of three synthetic chromosomes–synVI, synIII, and synIXR—into a single poly-synthetic strain.
RATIONALE
A host of Sc2.0 chromosomes, including synVI, have now been constructed in discrete strains. With debugging steps, where the number of bugs scales with chromosome length, all individual synthetic chromosomes have been shown to power yeast cells to near wild-type (WT) fitness. Testing the effects of Sc2.0 chromosome consolidation to uncover possible synthetic genetic interactions and/or perturbations of native cellular networks as the number of designer changes increases is the next major step for the Sc2.0 project.
RESULTS
SynVI was rapidly assembled using nine sequential steps of SwAP-In (switching auxotrophies progressively by integration), yielding a ~240-kb synthetic chromosome designed to Sc2.0 specifications. We observed partial silencing of the left- and rightmost genes on synVI, each newly positioned subtelomerically relative to their locations on native VI. This result suggests that consensus core X elements of Sc2.0 universal telomere caps are insufficient to fully buffer telomere position effects. The synVI strain displayed a growth defect characterized by an increased frequency of glycerol-negative colonies. The defect mapped to a synVI design feature in the essential PRE4 gene (YFR050C), encoding the β7 subunit of the 20S proteasome. Recoding 10 codons near the 3′ end of the PRE4 open reading frame (ORF) caused a ~twofold reduction in Pre4 protein level without affecting RNA abundance. Reverting the codons to the WT sequence corrected both the Pre4 protein level and the phenotype. We hypothesize that the formation of a stem loop involving recoded codons underlies reduced Pre4 protein level.

Sc2.0 chromosomes (synI to synXVI) are constructed individually in discrete strains and consolidated into poly-synthetic (poly-syn) strains by “endoreduplication intercross.” Consolidation of synVI with synthetic chromosomes III (synIII) and IXR (synIXR) yields a triple-synthetic (triple-syn) strain that is ~6% synthetic overall—with almost 70 kb deleted, including 20 tRNAs, and more than 12 kb recoded. Genome sequencing of double-synthetic (synIII synVI, synIII synIXR, synVI synIXR) and triple-syn (synIII synVI synIXR) cells indicates that suppressor mutations are not required to enable coexistence of Sc2.0 chromosomes. Phenotypic analysis revealed a slightly slower growth rate for the triple-syn strain only; the combined effect of tRNA deletions on different chromosomes might underlie this result. Transcriptome and proteome analyses indicate that cellular networks are largely unperturbed by the existence of multiple synthetic chromosomes in a single cell. However, a second bug on synVI was discovered through proteomic analysis and is associated with alteration of the HIS2 transcription start as a consequence of tRNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-syn strain “omics” analyses.
CONCLUSION
Analyses of phenotypes, transcriptomics, and proteomics of synVI and poly-syn strains reveal, in general, WT cell properties and the existence of rare bugs resulting from genome editing. Deletion of subtelomeres can lead to gene silencing, recoding deep within an ORF can yield a translational defect, and deletion of elements such as tRNA genes can lead to a complex transcriptional output. These results underscore the complementarity of transcriptomics and proteomics to identify bugs, the consequences of designer changes in Sc2.0 chromosomes. The consolidation of Sc2.0 designer chromosomes into a single strain appears to be exceptionally well tolerated by yeast. A predictable exception to this is the deletion of tRNAs, which will be restored on a separate neochromosome to avoid synthetic lethal genetic interactions between deleted tRNA genes as additional synthetic chromosomes are introduced.

Structured Abstract
INTRODUCTION
Although much effort has been devoted to studying yeast in the past few decades, our understanding of this model organism is still limited. Rapidly developing DNA synthesis techniques have made a “build-to-understand” approach feasible to reengineer on the genome scale. Here, we report on the completion of a 770-kilobase synthetic yeast chromosome II (synII). SynII was characterized using extensive Trans-Omics tests. Despite considerable sequence alterations, synII is virtually indistinguishable from wild type. However, an up-regulation of translational machinery was observed and can be reversed by restoring the transfer RNA (tRNA) gene copy number.
RATIONALE
Following the “design-build-test-debug” working loop, synII was successfully designed and constructed in vivo. Extensive Trans-Omics tests were conducted, including phenomics, transcriptomics, proteomics, metabolomics, chromosome segregation, and replication analyses. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium.
RESULTS
To efficiently construct megabase-long chromosomes, we developed an I-SceI–mediated strategy, which enables parallel integration of synthetic chromosome arms and reduced the overall integration time by 50% for synII. An I-SceI site is introduced for generating a double-strand break to promote targeted homologous recombination during mitotic growth. Despite hundreds of modifications introduced, there are still regions sharing substantial sequence similarity that might lead to undesirable meiotic recombinations when intercrossing the two semisynthetic chromosome arm strains. Induction of the I-SceI–mediated double-strand break is otherwise lethal and thus introduced a strong selective pressure for targeted homologous recombination. Since our strategy is designed to generate a markerless synII and leave the URA3 marker on the wild-type chromosome, we observed a tenfold increase in URA3-deficient colonies upon I-SceI induction, meaning that our strategy can greatly bias the crossover events toward the designated regions.

By incorporating comprehensive phenotyping approaches at multiple levels, we demonstrated that synII was capable of powering the growth of yeast indistinguishably from wild-type cells (see the figure), showing highly consistent biological processes comparable to the native strain. Meanwhile, we also noticed modest but potentially significant up-regulation of the translational machinery. The main alteration underlying this change in expression is the deletion of 13 tRNA genes.

A growth defect was observed in one very specific condition—high temperature (37°C) in medium with glycerol as a carbon source—where colony size was reduced significantly. We targeted and debugged this defect by two distinct approaches. The first approach involved phenotype screening of all intermediate strains followed by a complementation assay with wild-type sequences in the synthetic strain. By doing so, we identified a modification resulting from PCRTag recoding in TSC10, which is involved in regulation of the yeast high-osmolarity glycerol (HOG) response pathway. After replacement with wild-type TSC10, the defect was greatly mitigated. The other approach, debugging by SCRaMbLE, showed rearrangements in regions containing HOG regulation genes. Both approaches indicated that the defect is related to HOG response dysregulation. Thus, the phenotypic defect can be pinpointed and debugged through multiple alternative routes in the complex cellular interactome network.
CONCLUSION
We have demonstrated that synII segregates, replicates, and functions in a highly similar fashion compared with its wild-type counterpart. Furthermore, we believe that the iterative “design-build-test-debug” cycle methodology, established here, will facilitate progression of the Sc2.0 project in the face of the increasing synthetic genome complexity.

Structured Abstract
INTRODUCTION
Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.”
RATIONALE
Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies.
RESULTS
We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain.
CONCLUSION
Synthetic yeast chromosome X was chemically synthesized from scratch, a rigorous, incremental step toward complete synthesis of the whole yeast genome. Thousands of designer modifications in synX revealed extensive flexibility of the yeast genome. We developed an efficient mapping method, PoPM, to identify bugs during genome synthesis, generalizable to any watermarked synthetic chromosome, and several details of yeast biology were uncovered by debugging. Considering the numerous gene-associated PCRTags available in the synthetic chromosomes, PoPM may represent a powerful tool to map interesting phenotypes of mutated synthetic strains or even mutated wild-type strains to the relevant genes. It may also be useful to study yeast genetic interactions when an unexpected phenotype is generated by alterations in two or more genes, substantially expanding understanding of yeast genomic and cellular functions. The PoPM method is also likely to be useful for mapping phenotype(s) resulting from the genome SCRaMbLE system.

Structured Abstract
INTRODUCTION
The overall organization of budding yeast chromosomes is driven and regulated by four factors: (i) the tethering and clustering of centromeres at the spindle pole body; (ii) the loose tethering of telomeres at the nuclear envelope, where they form small, dynamic clusters; (iii) a single nucleolus in which the ribosomal DNA (rDNA) cluster is sequestered from other chromosomes; and (iv) chromosomal arm lengths. Hi-C, a genomic derivative of the chromosome conformation capture approach, quantifies the proximity of all DNA segments present in the nuclei of a cell population, unveiling the average multiscale organization of chromosomes in the nuclear space. We exploited Hi-C to investigate the trajectories of synthetic chromosomes within the Saccharomyces cerevisiae nucleus and compare them with their native counterparts.
RATIONALE
The Sc2.0 genome design specifies strong conservation of gene content and arrangement with respect to the native chromosomal sequence. However, synthetic chromosomes incorporate thousands of designer changes, notably the removal of transfer RNA genes and repeated sequences such as transposons and subtelomeric repeats to enhance stability. They also carry loxPsym sites, allowing for inducible genome SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) aimed at accelerating genomic plasticity. Whether these changes affect chromosome organization, DNA metabolism, and fitness is a critical question for completion of the Sc2.0 project. To address these questions, we used Hi-C to characterize the organization of synthetic chromosomes.
RESULTS
Comparison of synthetic chromosomes with native counterparts revealed no substantial changes, showing that the redesigned sequences, and especially the removal of repeated sequences, had little or no effect on average chromosome trajectories. Sc2.0 synthetic chromosomes have Hi-C contact maps with much smoother contact patterns than those of native chromosomes, especially in subtelomeric regions. This improved “mappability” results directly from the removal of repeated elements all along the length of the synthetic chromosomes. These observations highlight a conceptual advance enabled by bottom-up chromosome synthesis, which allows refinement of experimental systems to make complex questions easier to address. Despite the overall similarity, differences were observed in two instances. First, deletion of the HML and HMR silent mating-type cassettes on chromosome III led to a loss of their specific interaction. Second, repositioning the large array of rDNA repeats nearer to the centromere cluster forced substantial genome-wide conformational changes—for instance, inserting the array in the middle of the small right arm of chromosome III split the arm into two noninteracting regions. The nucleolus structure was then trapped in the middle between small and large chromosome arms, imposing a physical barrier between them.

In addition to describing the Sc2.0 chromosome organization, we also used Hi-C to identify chromosomal rearrangements resulting from SCRaMbLE experiments. Inducible recombination between the hundreds of loxPsym sites introduced into Sc2.0 chromosomes enables combinatorial rearrangements of the genome structure. Hi-C contact maps of two SCRaMbLE strains carrying synIII and synIXR chromosomes revealed a variety of cis events, including simple deletions, inversions, and duplications, as well as translocations, the latter event representing a class of trans SCRaMbLE rearrangements not previously observed.
CONCLUSION
This large data set is a resource that will be exploited in future studies exploring the power of the SCRaMbLE system. By investigating the trajectories of Sc2.0 chromosomes in the nuclear space, this work paves the way for future studies addressing the influence of genome-wide engineering approaches on essential features of living systems.

Perturbation of cellular processes is a prevailing approach to understanding biology. To better understand the complicated biology that defines bacterial shape, a sensitive, high-content platform was developed to detect multiple morphological defect phenotypes using microscopy. We examined morphological phenotypes across the Escherichia coli K-12 deletion (Keio) collection at the mid-exponential growth phase, revealing 111 deletions perturbing shape. Interestingly, 64% of these were uncharacterized mutants, illustrating the complex nature of shape maintenance and regulation in bacteria. To understand the roles these genes play in defining morphology, 53 mutants with knockouts resulting in abnormal cell shape were crossed with the Keio collection in high throughput, generating 1,373 synthetic lethal interactions across 1.7 million double deletion mutants. This analysis yielded a highly populated interaction network spanning and linking multiple phenotypes, with a preponderance of interactions involved in transport, oxidation-reduction, and metabolic processes.IMPORTANCE Genetic perturbations of cellular functions are a prevailing approach to understanding cell systems, which are increasingly being practiced in very high throughput. Here, we report a high-content microscopy platform tailored to bacteria, which probes the impact of genetic mutation on cell morphology. This has particular utility in revealing elusive and subtle morphological phenotypes associated with blocks in nonessential cellular functions. We report 111 nonessential mutations impacting E. coli morphology, with nearly half of those genes being poorly annotated or uncharacterized. Further, these genes appear to be tightly linked to transport or redox processes within the cell. The screening platform is simple and low cost and is broadly applicable to any bacterial genomic library or chemical collection. Indeed, this is a powerful tool in understanding the biology behind bacterial shape.