Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

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- Sce I–mediated strategy, which enables parallel integration of synthetic chromosome arms and reduced the overall integration time by 50% for synII. An I- Sce I 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- Sce I–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- Sce I 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. SynII characterization. ( A ) Cell cycle comparison between synII and BY4741 revealed by the percentage of cells with separated CEN2-GFP dots, metaphase spindles, and anaphase spindles. ( B ) Replication profiling of synII (red) and BY4741 (black) expressed as relative copy number by deep sequencing. ( C ) RNA sequencing analysis revealed that the significant up-regulation of translational machinery in synII is induced by the deletion of tRNA genes in synII. </jats:sec

Corrigendum to: The TianQin project: current progress on science and technology

In the originally published version, this manuscript included an error related to indicating the corresponding author within the author list. This has now been corrected online to reflect the fact that author Jun Luo is the corresponding author of the article

Ambiguity Function Analysis and Side Peaks Suppression of WiFi Signal for Passive Radar

WiFi (Wireless Fidelity) is widely deployed all the world. When it is utilized as external illuminaor in Passive Radar, its broadband singal make the high resolution of detection be obtained in both the range and Doppler domains. In this paper, the typical WiFi signal format and its characters are analyzed, then the theoretical signal model is setup. Based on the theory of bistatic passive radar, the relationship between typical IEEE 802.11 signals format and the characters of its Ambiguity Function (AF) is analyzed. Moreover, the position and amplitude of side peaks in time and frequency domain is analyzed and its causes from the signal structure is also discussed. In this paper, a method for suppressing the side peaks based on the correction of direct-path reference signal is proposed, therefore to avoid the false alarm brought in target detection caused by side peak interference. Experimental results valid the proposed signal processing method

Microstructure and properties of KNO₃- based water-soluble salt core strengthened by glass fiber

The KNO3-based water-soluble salt core was prepared using 70%KNO3-30%KCl (mole fraction) as a matrix material and glass fiber as a reinforcing material by stirring casting method. The effects of different glass fiber contents on the bending strength, impact toughness, water solubility rate and hygroscopic rate of the water-soluble salt core were compared and analyzed. The scanning electron microscopy and energy spectrometer were used to analyze the microstructure characteristics of the water-soluble salt core strengthened by glass fiber. The results show that with the increase of glass fiber content, the bending strength and impact toughness of the salt core are increased, and the water solubility rate and moisture absorption rate are gradually decreased. When the mass fraction of glass fiber is 30%, the salt core has the largest bending strength of (38.85±0.61) MPa and impact toughness with (2.13±0.1) kJ/m2, and the water solubility rate is still as high as (476.5±12.0) g/(min·m2), and the moisture absorption rate is (0.085±0.007)%. The microanalysis shows that the glass fibers are evenly distributed among the matrix of KNO3-based water-soluble salt core, which significantly refines the KCl primary phase. The average grain size of KCl primary phase is reduced from 57.89 μm to 24.13 μm, which is the main strengthening mechanism of the salt core. The crack will deflect when encountering glass fibers during the crack propagation, and the fiber pull-out is also observed, which is the main toughening mechanism of the salt core

Molecular identification of a root apical cell-specific and stress-responsive enhancer from an Arabidopsis enhancer trap line

Abstract Background Plant root apex is the major part to direct the root growth and development by responding to various signals/cues from internal and soil environments. To study and understand root system biology particularly at a molecular and cellular level, an Arabidopsis T-DNA insertional enhancer trap line J3411 expressing reporters (GFP) only in the root tip was adopted in this study to isolate a DNA fragment. Results Using nested PCR, DNA sequencing and sequence homology search, the T-DNA insertion site(s) and its flanking genes were characterised in J3411 line. Subsequently, a 2000 bp plant DNA-fragment (Ertip1) upstream of the insert position of the coding T-DNA was in silico analysed, revealing certain putative promoter/enhancer cis-regulatory elements. Cloning and transformation of this DNA fragment and its truncated segments tagged with or without 35S minimal promoter (35Smini), all of which were fused with a GFP or GUS reporter, allowed to detect GFP and GUS expression mediated only by Ertip1 + 35mini (PErtip1+35Smini) specifically in the Arabidopsis root tip region. The PErtip1+35Smini activity was further tested to be strong and stable under many different growth conditions but suppressed by cold, salt, alkaline pH and higher ammonium and phosphorus. Conclusion This work describes a promising strategy to isolate a tissue-/cell-specific enhancer sequence from the enhancer trap lines, which are publically available. The reported synthetic promoter i.e. PErtip1+35Smini may provide a valuable and potent molecular-tool for comprehensive investigation of a gene function related to root growth and development as well as molecular engineering of root-architectural formation aiming to improve plant growth

Satellite breakup behaviors and model under the hypervelocity impact and explosion: A review

The primary causes of satellite breakups are hypervelocity impact and explosion, the research on satellite breakup can be used not only to evaluate the influence of breakup event on the space environment, but also to trace whether the satellite has been deliberately attacked. It is of great significance in both civil and military aspects. The study of satellite breakup behaviors and model is reviewed to summarize the research progress and insufficiency in recent decades, including the satellite breakup experiment, measurement and characterization of fragments, distribution characteristics of breakup fragments, satellite breakup model, etc. The classical studies are introduced in detail, and the limitations of the current research are pointed out. According to the current research results, the contemporary challenges and future directions for satellite breakup study are presented. The research on satellite breakup is developing in two directions: the miniaturization of satellite size and the complexity of satellite component. The study on satellite breakup needs to be explored and deepened on improving the experimental launch speed, expanding the model application range and breakup revealing the results under combined effect of impact and explosion