BioPartsBuilder: a synthetic biology tool for combinatorial assembly of biological parts

Abstract Summary: Combinatorial assembly of DNA elements is an efficient method for building large-scale synthetic pathways from standardized, reusable components. These methods are particularly useful because they enable assembly of multiple DNA fragments in one reaction, at the cost of requiring that each fragment satisfies design constraints. We developed BioPartsBuilder as a biologist-friendly web tool to design biological parts that are compatible with DNA combinatorial assembly methods, such as Golden Gate and related methods. It retrieves biological sequences, enforces compliance with assembly design standards and provides a fabrication plan for each fragment. Availability and implementation: BioPartsBuilder is accessible at http://public.biopartsbuilder.org and an Amazon Web Services image is available from the AWS Market Place (AMI ID: ami-508acf38). Source code is released under the MIT license, and available for download at https://github.com/baderzone/biopartsbuilder. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.</jats:p

Context-dependent neocentromere activity in synthetic yeast chromosome VIII

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae. With the completion of synthetic chromosome VIII (synVIII) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII, we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (~500 bp) of the CDEIII-proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality </p

Synthetic chromosome fusion: Effects on mitotic and meiotic genome structure and function

We designed and synthesized synI, which is ~21.6% shorter than native chrI, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance and is now attached to synIII, yielding the first synthetic yeast fusion chromosome. Additional fusion chromosomes were constructed to study nuclear function. ChrIII-I and chrIX-III-I fusion chromosomes have twisted structures, which depend on silencing protein Sir3. As a smaller chromosome, chrI also faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of the crossover-promoting protein Red1. These effects extend over 100 kb and promote disproportionate Red1 enrichment, and thus crossover potential, on small chromosomes like chrI. These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems  </p

KARYOTYPE ENGINEERING: SYNTHESIS AND FUSION OF YEAST CHROMOSOMES

As part of the Sc2.0 project, we designed and synthesized synthetic chromosome I. The total length of synI is ~21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype balance. We used a robust CRISPR-Cas9 method to fuse chromosome I to chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew as well as wild type so we decided to attach synI to synIII. Through investigation of three-dimensional structures of fusion chromosome strains, a strange loop and a twisted complex structure were formed in chrIII-I and chrIX-III-I fusion chromosomes, which are dependent on the presence of silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. In meiosis, we used these fusion chromosomes to show that axial element Red1 binding is not strictly chromosome size dependent even though Red1 binding is enriched on the three small chromosomes in wild-type yeast and we discovered an unexpected role for centromeres in Red1 binding. Using this CRISPR-Cas9 method, we have successfully fused yeast chromosomes, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from n=16 to n=2. A strain carrying only two ~6 Mb long chromosomes exhibited only modest transcriptomic changes and strikingly, grows without major defects compared to the n=16 strain. In heterotypic crosses (n<16 X n=16), two trends were noted. As n dropped below 16, spore viability decreased dramatically, reaching <10% at n=12 X n=16. As n decreased further, yeast sporulation was arrested, with drastically reduced full tetrad formation detected in the n=8 X n=16 cross, with <1% spore viability, from which no viable spores could be recovered. However, homotypic crosses between pairs of n=8, 4 and 2 strains gave excellent sporulation and spore viability. These results indicate that as few as 8 chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates reduction in chromosome number surprisingly well, providing a dramatic new example of the remarkable robustness of the yeast genome to change

Karyotype engineering reveals spatio-temporal control of replication firing and gene contacts

International audienceEukaryotic genomes vary in terms of size, chromosome number, and genetic complexity. Their temporal organization is complex, reflecting coordination between DNA folding and function. Here, we used fused karyotypes of budding yeast to characterize the effects of chromosome length on nuclear architecture. We found that size-matched megachromosomes expand to occupy a larger fraction of the enlarged nucleus. Hi-C maps reveal changes in the three-dimensional structure corresponding to inactivated centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication, highlighting a functional correlation between genome organization and replication timing. Repositioning of former telomere-proximal regions on chromosome arms exposed a subset of contacts between flocculin genes. Chromatin reorganization of megachromosomes during cell division remained unperturbed, and it revealed that centromere-rDNA contacts in anaphase, extending over 0.3 Mb on wild-type chromosome, cannot exceed ∼1.7 Mb. Our results highlight the relevance of engineered karyotypes to unveiling relationships between genome organization and functio

Karyotype engineering reveals spatio-temporal control of replication firing and gene contacts

International audienceEukaryotic genomes vary in terms of size, chromosome number, and genetic complexity. Their temporal organization is complex, reflecting coordination between DNA folding and function. Here, we used fused karyotypes of budding yeast to characterize the effects of chromosome length on nuclear architecture. We found that size-matched megachromosomes expand to occupy a larger fraction of the enlarged nucleus. Hi-C maps reveal changes in the three-dimensional structure corresponding to inactivated centromeres and telomeres. De-clustering of inactive centromeres results in their loss of early replication, highlighting a functional correlation between genome organization and replication timing. Repositioning of former telomere-proximal regions on chromosome arms exposed a subset of contacts between flocculin genes. Chromatin reorganization of megachromosomes during cell division remained unperturbed, and it revealed that centromere-rDNA contacts in anaphase, extending over 0.3 Mb on wild-type chromosome, cannot exceed ∼1.7 Mb. Our results highlight the relevance of engineered karyotypes to unveiling relationships between genome organization and functio

Ulinastatin Ameliorates IL-1β-Induced Cell Dysfunction in Human Nucleus Pulposus Cells via Nrf2/NF-κB Pathway

Low back pain (LBP) has been a wide public health concern worldwide. Among the pathogenic factors, intervertebral disc degeneration (IDD) has been one of the primary contributors to LBP. IDD correlates closely with inflammatory response and oxidative stress, involving a variety of inflammation-related cytokines, such as interleukin 1 beta (IL-1β), which could result in local inflammatory environment. Ulinastatin (UTI) is a kind of acidic protein extracted from human urine, which inhibits the release of tumor necrosis factor alpha (TNF-α) and other inflammatory factors to protect organs from inflammatory damage. However, whether this protective effect of UTI on human nucleus pulposus (NP) exists, and how UTI affects the biological behaviors of human NP cells during IDD remain elusive. In this current study, we revealed that UTI could improve the viability of NP cells and promote the proliferation of NP cells. Additionally, UTI could protect human NP cells via ameliorating IL-1β-induced apoptosis, inflammatory response, oxidative stress, and extracellular matrix (ECM) degradation. Molecular mechanism analysis suggested that the protective effect from UTI on IL-1β-treated NP cells were through activating nuclear factor- (erythroid-derived 2-) like 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway and the suppression of NF-κB signaling pathway. Therefore, UTI may be a promising therapeutic medicine to ameliorate IDD

AIEE-Active and Electrochromic Bifunctional Polymer and a Device Composed thereof Synchronously Achieve Electrochemical Fluorescence Switching and Electrochromic Switching

A novel alternating polymer, ProDOT-TPE, with aggregation-enhanced fluorescent emission and electrochromic properties based on thiophene and tetraphenylethene derivatives was designed, synthesized, and characterized. The polymer displays weak photoluminescence in tetrahydrofuran, but its corresponding film prepared by spray-coating exhibits yellow-green fluorescent light at 540 nm. The color of the polymer film could be switched from bright yellow to navy blue by applying a relatively low voltage. An electrochromic device (ECD) of the polymer was fabricated that differs from common ECDs because both its color and fluorescent state could be synchronously switched by an applied voltage, making the polymer a unique candidate for electrochemical fluorescence and electrochromic applications

Anthropogenic heat due to energy consumption contributes to cooler and wetter summers in Southwest China

Anthropogenic heat release (AHR) is the release of heat generated by anthropogenic energy consumption. The regional mean Anthropogenic heat release flux in Southwest China (SWC) grew quickly from 0.06 Wm-2 in 1992 to a peak of 0.37Wm-2 in 2019. This study examines the climatic effects and feedbacks of Anthropogenic heat release due to energy consumption in Southwest China during the boreal summer using simulations from the Community Earth System Model. The modeling results show that Anthropogenic heat release impacts on the lower-troposphere stability and affects large-scale atmospheric circulation in Southwest China, which transports more water vapor and consequently increases the humidity and low cloud cover in Southwest China. This effect impacts the energy balance at the surface by reducing the amount of incoming shortwave radiation that reaches the ground. Anthropogenic heat release decreases the average 2-m air temperature in Southwest China by 0.10 ± 0.01K (1σ uncertainty) and also decreases the minimum and maximum air temperatures in Southwest China as well. Anthropogenic heat release contributes to cooler and wetter summers in Southwest China. The results show that Anthropogenic heat release is a non- negligible factor that impacts the climate of Southwest China. This study improves our understanding of the climate change resulting from human activities in Southwest China

RADOM, an Efficient <i>In Vivo</i> Method for Assembling Designed DNA Fragments up to 10 kb Long in <i>Saccharomyces cerevisiae</i>

We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects