Search

C/SiC composite has excellent performance, but their damage patterns cannot be effectively detected, and it is tough to obtain a large amount of label data. Therefore, an autonomous damage image recognition based on transfer learning is proposed in this paper. A C/SiC composite damage image data set is collected from experiments and papers, including fiber pullout, matrix cracking, interface debonding, and fiber breakage images. Pre-trained DenseNet121 was transferred to train and classify this data set. Pre-trained AlexNet and other four networks are selected for comparison. The results of experiment illustrate that the accuracy of directly using the DenseNet121 model is 70.536%, and the accuracy of the transferring pre-trained AlexNet, VGG16, ResNet18, and MobileNet-v2 on the validation set is 96.429%, 84.598%, 94.420%, 94.866%, respectively. The accuracy in this paper is 97.098%. As the number of damage images of C/SiC composite is limited, the recognition accuracy of the method is significantly improved.

An increasing number of Bacillus strains have been identified, and the removal capacity of zearalenone (ZEN) was determined; however, they failed to reveal the detoxification mechanism and transformation product. Here, Bacillus subtilis Y816, which could transform 40 mg/L of ZEN within 7 h of fermentation, was identified and studied. First, the biotransformation products of ZEN and 17-β-estradiol (E2) were identified as ZEN-14-phosphate and E2-3-phosphate by HPLC-TOF-MS and NMR, respectively. An intracellular zearalenone phosphotransferase (ZPH) was found through transcriptome sequencing analysis of B. subtilis Y816. The phosphorylated reaction conditions of ZEN by ZPH were further revealed in this work. Furthermore, the phosphorylated conjugates showed reduced estrogenic toxicity compared with their original substances (ZEN and α/β-zearalenol) using an engineered yeast biosensor system. The first report on the phosphorylated conjugated mode of ZEN in B. subtilis Y816 will inspire new perspectives on the biotransformation of ZEN in Bacillus strains.

Mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for treating many diseases. However, their proliferation and therapeutic abilities decline during the aging process and disease development. Therefore, fetal MSCs derived from the umbilical cord (UC) attract more attention. Storing and delivering the UC is one critical step for efficient MSC isolation. Although the culture medium-based solution is suitable for UC storage, it is not feasible for large-scale preparation because of its high price. Thus, we demonstrate here that a simple solution containing a pH buffering reagent, calcium, magnesium and glucose could be used as a cost-effective storage solution for UC delivery and efficient MSC isolation.

Objectives Mesenchymal stem cells (MSCs) have been intensively investigated as to their therapeutic potentials. However, the full chemical-defined medium supporting the isolation and expansion of human MSCs has not been developed yet. Materials and methods Here, we developed the full chemical-defined medium, NBVbe medium, via RNA sequencing, bioinformatic analysis, and growth factor screening. Results The NBVbe medium contains N2B27 medium with the BSA (bovine serum albumin) replaced by the recombinant human albumin, bFGF (basic fibroblast growth factor), vitamin C, and EGF (epidermal growth factor). The NBVbe medium could support the isolation and expansion of human MSCs from the umbilical cords. Conclusions The full chemical-defined medium supporting the isolation and expansion of human MSCs has been developed. This would be helpful for further optimization of the MSC medium, their clinical applications, and molecular characterization.

Background pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli–B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. Results The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. Conclusion The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

International audience; Organic mudstone can be a high-quality source rock in petroleum basins. The accumulation patterns of various types of mudstone can also serve as a useful record of paleogeography. Due to the impact of tectonic movements, the East China Sea Basin (ECSB) saw its deposition setting transform from closed-lacustrine to open marine settings. This provides an opportunity to explore organic matter accumulation patterns within such a transformation setting. This study classified the constituent mudstones according to their differences in composition and sedimentary structure as follows: coaly mudstone or carbonaceous mudstone, calcareous mudstone, clayey mudstone, and siliceous mudstone. Considering the different sedimentary structures, siliceous mudstone can be subdivided into laminated, wavy, and slumped mudstones. Using integrated multiple indices of the paleo-environment, which includes clay mineral content, trace mineral content, and micro-paleontology assemblages, we reconstructed the paleography setting during the Paleocene and inferred that during this time, the ECSB was characterized by sedimentary differentiation. We discerned two different organic mudstone accumulation patterns by combining the results from rock-eval pyrolysis and paleo-environmental indices: 1) A closed-lacustrine mudstone accumulation pattern characterized by abundant sediment supply and dominated by allogenous mudstone formation by the flocculation process. The fresh to slightly salty water environment provided adequate nutrition to microorganisms and an appropriate temperature led to the increased accumulation of type II1 and II2 kerogen. 2) An open-marine mudstone accumulation pattern which revealed a sediment-starved setting. The flocculation process and fine-graded turbidities were the main processes leading to mudstone accumulation. More plants were concentrated in shallow water with declining salinity, leading to peat and mass carbon mudstone accumulation.

A novel Escherichia coli efficient expression system had been constructed in our previous study. The system was based on the overexpression of endogenous genes prpD and malK to enhance the expression of exogenous genes. In this study, a general regulatory mechanism of prpD and malK was first revealed through transcriptome analysis and many experimental verifications. We surprisingly proved that overexpression of malK could up-regulate the expression of prpD and propanoate metabolism, which leads to increased expression of exogenous genes. More importantly, the overexpression of prpD or malK could arouse a complex set of pyruvate-centered metabolic networks that mainly increase the energy supply (ATP), by-product recycling (acetate), and amino acids for the efficient expression of exogenous genes. This novel theory for promoting the efficient expression of exogenous genes will be useful in a wide range of fields. It also opens up a new perspective on the regulation of metabolism in E. coli cells.

Colloidal lithography provides a rapid and low-cost approach to construct 2D periodic surface nanostructures. However, an impressive demonstration to prepare large-area colloidal template is still missing. Here, a high-efficient and flexible technique is proposed to fabricate self-assembly monolayers consisting of orderly-packed polystyrene spheres at air/water interface via ultrasonic spray. This "non-contact" technique exhibits great advantages in terms of scalability and adaptability due to its renitent interface dynamic balance. More importantly, this technique is not only competent for self-assembly of single-sized polystyrene spheres, but also for binary polystyrene spheres, completely reversing the current hard situation of preparing large-area self-assembly monolayers. As a representative application, hexagonal-packed silver-coated silicon nanorods array (Si-NRs@Ag) is developed as an ultrasensitive surface-enhanced Raman scattering (SERS) substrate with very low limit-of-detection for selective detection of explosive 2,4,6-trinitrotoluene down to femtomolar (10

Bacillus subtilis has been widely used as a prokaryotic host for the secretory expression of heterologous proteins. In this study, a pullulanase (PulA) from Anoxybacillus sp. LM18-11 was firstly identified to be expressed in Bacillus subtilis 1A751 through non-classical secretion pathway. Results showed that both the N- and C-terminal regions of PulA were essential for its soluble expression. To explore its specific structural basis of secretion in B. subtilis, we revealed a hydrophobic motif A501-H507 which is vital for the secretion of the whole protein of PulA. Through a series of site-specific mutagenesis, the triple-sites mutants R503E/I506E/H507E and R503E/I506Y/H507E showed the highest extracellular activity (160.07 U/mL) and total activity (243.37 U/mL) which was 1.71 times and 1.55 times higher than those of PulA. The highest secretion rate of mutant I506E/H507E was more than 50% which was 34.72% higher comparing with that of PulA. The glutamic acid substitution on these three key surface sites which decreased the surface hydrophobicity of that region was confirmed to be beneficial to improve the secretory expression of PulA. This novel discovery for the secretory expression of PulA in B. subtilis would make a new perspective on regulating a kind of non-classical secretion in B. subtilis.

Objectives Myocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor-like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus-based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene-free approaches. Materials and methods Four transgene-free approaches for somatic reprogramming, including episome, minicircle, self-replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations. Results The self-replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C-X-C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach. Conclusion Thus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.

Objectives Myocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor‐like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus‐based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene‐free approaches. Materials and Methods Four transgene‐free approaches for somatic reprogramming, including episome, minicircle, self‐replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations. Results The self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C‐X‐C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach. Conclusion Thus, the optimized strategy for iCS generation is safer and has more therapeutic potentials., Previously, we and others have successfully reprogrammed mouse fibroblast cells into cardiac progenitor‐like cells. However, several issues should be further addressed, including the reprogramming strategy and delivery approach for myocardial infarction (MI) treatment. In the current study, we screened several transgene‐free approaches for somatic reprogramming. Our data showed the self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. Then, the CXCR4+ (C‐X‐C chemokine receptor 4) subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a more safer delivery approach. Thus, the optimized strategy for iCS generation is much safer and has more therapeutic potentials.

Osteosarcoma (OS) is the most common primary malignant tumor of bone. It is a common phenomenon that osteosarcoma cells have a hypoxic microenvironment. Hypoxia can dedifferentiate cells of several malignant tumor types into stem cell-like phenotypes. However, the role of hypoxia in stemness induction and the expression of cancer stem cell (CSC) markers in human osteosarcoma cells has not been reported. The present study examined the effects of hypoxia on stem-like cells in the human osteosarcoma MNNG/HOS cells. Under the incubation with 1% oxygen, the expression of CSCs markers (Oct-4, Nanog and CD133) in MNNG/HOS cells were increased. Moreover, MNNG/HOS cells cultured under hypoxic conditions were more likely to proliferate into spheres and resulted in larger xenograft tumor. Hypoxia also increased the mRNA and protein levels of hypoxia-inducible factor (HIF)-1α. Then rapamycin was used, which has been shown to lower HIF-1α protein level, to inhibit the hypoxic response. Rapamycin suppressed the expression of HIF-1α protein and CSCs markers (Oct4, Nanog and CD133) in MNNG/HOS cells. In addition, pretreatment with rapamycin reduced the efficiency of MNNG/HOS cells in forming spheres and xenograft tumors. The results demonstrated that hypoxia (1% oxygen) can dedifferentiate some of the MNNG/HOS cells into stem cell-like phenotypes, and that the mTOR signaling pathway participates in this process via regulating the expression of HIF-1α protein.

International audience; For complex convergence movements between the Pacific Plate and Eurasian Plate since the Cenozoic, the East China Sea Basin (ECSB) presents a three-stage rifting movement: initial rifting, rifting development, and structural reversal stages. The ECSB changed from a closed lacustrine to an open marine source-to-sink (S2S) system and then returned to a semiopen bay S2S system. Recently, research has attracted more attention to the relationship between the depositional setting evolution and the corresponding source-to-sink patterns. This study determines that the early Paleocene was dominated by a closed lacustrine setting according to water salinities based on trace element and micropalaeontological assemblages. The middle Paleocene had an open marine setting, and the late Paleocene and Eocene had semiopen bay settings. In addition, we rebuild the provenance area of the S2S system with detrital zircon Usingle bondPb data and heavy mineral assemblage data to interpret the provenance from seismic profiles. During the early Paleocene, proximal sediments dominated the western depression zone of the East China Sea (WDZ), which was isolated by three sags. Integrated from core, well logging, and grain size data, the sediments in the routing system migrated to the prograding delta or alluvial fan. With statistics of the S2S parameters on the early Paleocene strata, sediment supply, average terrain height, and volume of the S2S system are analyzed from a bubble chart, which indicates that the average terrain height had a positive correlation with the S2S volume. For the middle Paleocene, more sediments from the Zhe-Min Caledonian fault belts on the western side distally migrated to the drainage basin and were deposited as tidally dominated deltas, reflecting an increasing reworking process. The statistical results show that the sediment supply was a dominant factor in the S2S system. As the regression proceeded, the S2S system developed in a semiopen bay setting in the late Paleocene and Eocene. A wave-dominated delta existed on the western side of the WDZ, and the eastern side developed subaqueous deltas. Moreover, we note that the sediment supply and average terrain height were important factors in developing the S2S system

International audience; Pearl River Mouth Basin (PRMB) is a typical episodically rifting basin, and its sediment records exhibit a multistage infilling process. To build the coupling relationship between the spatial variability of tectonic movement and sediment dispersal pattern, we character the source-to-sink system distribution within continental margin and analysis their dominated factors with quantitative statistical methods. First, we identify the faults and calculate the rifting rate by interpreting the two-dimensional (2D) seismic data and zircon or apatite fission-track data (ZFT/AFT). Accordingly, based on the various trending of the rifting rate evolution, we classified rifting process episodically into three patterns: accelerated rifting decelerated rifting, and uniform rifting. Then, we integrated the data available on detrital zircon U–Pb, rare earth element, and heavy mineral assemblage to identify the responding S2S components associated with the above various rifting processes. Results show that in the first tectonic stage during the early Eocene, the sediments were mainly sourced from an intrabasin provenance area, whereas in the late Eocene to the Oligocene, the paleo-Pearl River provided the sediments to the sink area. We restore the facies association of the sink area by studying the core samples, electric well logs, and three-dimensional (3D) seismic data. During the accelerated and uniform rifting processes, coarse-grained deposited along the boundary faults, which includes the fan delta and subaqueous fans. During decelerated rifting, the depositional environment was dominated by braided channels, meandering channels, and tide-influenced channel accretion. Based on the quantitative analysis the relationship between the grain size of the sediment in each S2S segment and the rifting rate, more fine-grained components were preserved in the decelerated rifting system, while coarse-grained sediments fill the accelerated rifting depression and uniform rifting components. Furthermore, the sediment supply and relative height can influence the volume of the S2S system in the uniform rifting system. In the decelerated rifting basin, the sediment supply is the major factor associated with the development of S2S, whereas the relative height is the major factor associated with the accelerated rifting basin.

A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo- was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo- achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.

A high heterologous expression of an alkaline pectate lyase (APL) pelNK93I in E. coli was obtained through optimizing the lactose feeding and fed-batch fermentation. The highest soluble APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 10,181 U/mL which is the highest level so far. On this basis, to improve the extracellular yield of APL, optimized glycine feeding was used to achieve elevated extracellular production of pelNK93I. The highest extracellular APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 6357 U/mL which was also relatively higher than that in previous reports. The final productivity of APL was 282.8 U/mL/h in the fermentation of E. coli BL21 (pET22b-pelNK93I) in a 10 L fermenter. Thus the current study has provided a cost-effective method for the over-expression and preparation of alkaline pectate lyase pelNK93I for its industrial applications. Moreover, pelNK93I (4 U/mL) used for bioscouring increased cottonseed husk removal and radial capillary effect of cotton fabric by 37.63% and 47.06%, respectively, making it a promising enzyme in green textile technology.

Direct ink writing promotes ink formulation design gradually become a hotspot in the application research of nanothermite. In order to obtain an energetic ink with a certain mechanical strength and formability, it is necessary to compound the energetic material with an inert binder. The inert component will not only reduce the sensitivity and energy release efficiency of energetic systems, but also affect its application in micro ignition devices. Based on the thermal gelation behavior of hydroxypropyl methylcellulose (HPMC), the nAl/pCuO/HPMC ink was prepared by HPMC, nano aluminum powder (nAl), and sheet-like porous CuO (pCuO). It has found out that the energetic ink has a favorable printing accuracy when the HPMC content is 7 wt%. To compare the influence from the size and morphology of copper oxide and aluminum on ink printing and reaction performances, the mAl/pCuO/HPMC, nAl/nCuO/HPMC and nAl/mCuO/HPMC inks have been prepared using the same way. Due to the large particle size of the components, nAl/mCuO/HPMC cannot be printed continuously during the printing process. For the mAl/pCuO/HPMC, it cannot form self-sustaining combustion on the glass substrate when the ink is a single layer. The activation energy results show that nAl/pCuO/HPMC has the smallest value (68.39 kJ/mol). The calculated burning rate of nAl/pCuO/HPMC (32.56 cm/s) is about 6 times than that of nAl/nCuO/HPMC system (5.28 cm/s). The ignition performance test shows that only the nAl/pCuO/HPMC system can be successfully ignited by a SCB micro igniter when the HPMC content is 7 wt%. Meanwhile, the pressure test indicates that the nAl/pCuO/HPMC system (7 wt% HPMC, Ф=1.4) has the shortest pressure rising time (46.7 μs), but the largest pressurization rate (1661.7 GPa/s).

A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.

Background: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli - B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence ( ori ) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1~pUC980-8) containing all possible insertion sites and directions were constructed.Results: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN , an alkaline protease spro1 and a pullulanase gene pulA11 , respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were upto 5200 U/mL, 21537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports.Conclusion: In this work we can conclude that the optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis . And the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

Background/Aims: The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA). Methods: Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy. Results: The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification. Conclusions: Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.