Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes. </p

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes. </p

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

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 incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, 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 new opportunities to directly test hypotheses surrounding these essential non-coding RNAs

Design, construction, and functional characterization of a tRNA neochromosome in yeast

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

Design, construction, and functional characterization of a tRNA neochromosome in yeast

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

Investigation of the Thermal Degradation of SBS Polymer in Long-Term Aged Asphalt Binder Using Confocal Laser Scanning Microscopy (CLSM)

Styrene–butadiene–styrene (SBS) polymer is extensively employed for asphalt pavement construction, and its degradation significantly damages the durability of asphalt concrete. However, the effect of aging protocols on the degradation of SBS polymer in asphalt binder has not been thoroughly investigated. In this study, confocal laser scanning microscopy (CLSM) was applied to characterize the change in morphology with SBS polymer degradation. Various aging protocols were considered, including accelerated aging processes in laboratory- and field-aged samples from three highway sections with different in-service periods. Scanned images of the polymer phase in the 2D plane at different depths were processed and further reconstructed in three dimensions. Furthermore, the three-dimensional polymer morphology indices derived from the semi-quantitative analysis of the images were correlated with the rheological indices. The results show that the polymer particles change from a relatively large ellipsoidal shape to a relatively small spherical shape as aging proceeds. The increase in aging temperature appears to accelerate the degradation of the polymer at the same rheological level. The effect of the laboratory aging method on the polymer was more pronounced during the early stages of aging compared to that in the field aging process

Investigation of the Thermal Degradation of SBS Polymer in Long-Term Aged Asphalt Binder Using Confocal Laser Scanning Microscopy (CLSM)

Styrene&ndash;butadiene&ndash;styrene (SBS) polymer is extensively employed for asphalt pavement construction, and its degradation significantly damages the durability of asphalt concrete. However, the effect of aging protocols on the degradation of SBS polymer in asphalt binder has not been thoroughly investigated. In this study, confocal laser scanning microscopy (CLSM) was applied to characterize the change in morphology with SBS polymer degradation. Various aging protocols were considered, including accelerated aging processes in laboratory- and field-aged samples from three highway sections with different in-service periods. Scanned images of the polymer phase in the 2D plane at different depths were processed and further reconstructed in three dimensions. Furthermore, the three-dimensional polymer morphology indices derived from the semi-quantitative analysis of the images were correlated with the rheological indices. The results show that the polymer particles change from a relatively large ellipsoidal shape to a relatively small spherical shape as aging proceeds. The increase in aging temperature appears to accelerate the degradation of the polymer at the same rheological level. The effect of the laboratory aging method on the polymer was more pronounced during the early stages of aging compared to that in the field aging process

Experimental Study on Seismic Behavior of Masonry Walls Strengthened by Reinforced Mortar Cross Strips

Due to the poor seismic performance, strengthening of masonry structures is always a significant problem worthy to study. It has been proven that the bearing capacity of existing masonry buildings can be enhanced greatly with efficient strengthening measures. An experimental program was conducted to investigate seismic performance of un-reinforced masonry (URM) walls strengthened by reinforced mortar (RM) cross strips. Eleven walls were tested under horizontal low-cyclic load, simultaneously with a vertical constant load on the top face. Three URM walls were tested as reference. The other eight walls were externally strengthened with 40 and 60 mm thick of RM cross strips on one or both faces. Test results showed that externally strengthening with RM cross strips was an efficient way to enhance the seismic performance of URM walls. The failure modes were divided into shear failure and shear-compression failure. All the tested walls did not collapse until the test ended, while many diagonal cracks and few vertical cracks appeared on mortar strips. After strengthening, the shear capacity of the strengthened walls increased by at least 38.2%, and the reinforcement ratio was noted to be the key factor to influence the shear capacity with positive correlation. Besides, RM cross strips did improve deformation capacity greatly

Experimental investigation and analytical prediction on bond behaviour of CFRP-to-concrete interface with FRP anchors

Hybrid bonded technology with fiber-reinforced polymer (FRP) anchors can effectively improve the load capacity of carbon fiber-reinforced polymer (CFRP)-to-concrete interface. To investigate the reinforcement effect of FRP anchors, tensile shear tests of seventeen CFRP-concrete bonded joints with additional FRP anchors were conducted in this paper. The effects of the construction sequence, anchor position, and type of FRP anchor were considered in the experimental investigation. It was found that bonding CFRP sheet before installing FRP anchor and construction simultaneously could ensure the integrity of FRP anchor and provide a better anchor effect. The ultimate load-carrying capacity was increased with the increasing of plate length after anchor, that is the effect of anchor position. From the results of specimens with different types of FRP anchors at the same location, the FRP anchor composed of multiple small anchors can take effect earlier. By fitting the test results, the exponential bond-slip model for the CFRP-to-concrete interface and the elastic constitutive model for the FRP anchor were obtained. Further, substituting the two constitutive models, analytic theoretical models for load-slip curves and strain distribution were derived to predict the bond behavior of FRP anchored CFRP-to-concrete interface in this paper. The comparison between the prediction results and experimental data shows the good predictive accuracy of these analytical models

Full-Scale Pore Structure and Fractal Dimension of the Longmaxi Shale from the Southern Sichuan Basin: Investigations Using FE-SEM, Gas Adsorption and Mercury Intrusion Porosimetry

Pore structure determines the gas occurrence and storage properties of gas shale and is a vital element for reservoir evaluation and shale gas resources assessment. Field emission scanning electron microscopy (FE-SEM), high-pressure mercury intrusion porosimetry (HMIP), and low-pressure N2/CO2 adsorption were used to qualitatively and quantitatively characterize full-scale pore structure of Longmaxi (LM) shale from the southern Sichuan Basin. Fractal dimension and its controlling factors were also discussed in our study. Longmaxi shale mainly developed organic matter (OM) pores, interparticle pores, intraparticle pores, and microfracture, of which the OM pores dominated the pore system. The pore diameters are mainly distributed in the ranges of 0.4&ndash;0.7 nm, 2&ndash;20 nm and 40&ndash;200 &mu;m. Micro-, meso- and macropores contribute 24%, 57% and 19% of the total pore volume (PV), respectively, and 64.5%, 34.6%, and 0.9% of the total specific surface area (SSA). Organic matter and clay minerals have a positive contribution to pore development. While high brittle mineral content can inhibit shale pore development. The fractal dimensions D1 and D2 which represents the roughness of the shale surface and irregularity of the space structure, respectively, are calculated based on N2 desorption data. The value of D1 is in the range of 2.6480&ndash;2.7334 (average of 2.6857), D2 is in the range of 2.8924&ndash;2.9439 (average of 2.9229), which indicates that Longmaxi shales have a rather irregular pore morphology as well as complex pore structure. Both PV and SSA positively correlated with fractal dimensions D1 and D2. The fractal dimension D1 decreases with increasing average pore diameter, while D2 is on the contrary. These results suggest that the small pores have a higher roughness surface, while the larger pores have a more complex spatial structure. The fractal dimensions of shale are jointly controlled by OM, clays and brittle minerals. The TOC content is the key factor which has a positive correlation with the fractal dimension. Clay minerals have a negative influence on fractal dimension D1, and positive influence D2, while brittle minerals show an opposite effect compared with clay minerals