PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in <i>Escherichia coli</i>

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli

Sintering Temperature Induced Evolution of Microstructures and Enhanced Electrochemical Performances: Sol-Gel Derived LiFe(MoO4)2 Microcrystals as a Promising Anode Material for Lithium-Ion Batteries

A facile sol-gel process was used for synthesis of LiFe(MoO4)2 microcrystals. The effects of sintering temperature on the microstructures and electrochemical performances of the as-synthesized samples were systematically investigated through XRD, SEM and electrochemical performance characterization. When sintered at 650°C, the obtained LiFe(MoO4)2 microcrystals show regular shape and uniform size distribution with mean size of 1–2 μm. At the lower temperature (600°C), the obtained LiFe(MoO4)2 microcrystals possess relative inferior crystallinity, irregular morphology and vague grain boundary. At the higher temperatures (680 and 700°C), the obtained LiFe(MoO4)2 microcrystals are larger and thicker particles. The electrochemical results demonstrate that the optimized LiFe(MoO4)2 microcrystals (650°C) can deliver a high discharge specific capacity of 925 mAh g−1 even at a current rate of 1 C (1,050 mA g−1) after 500 cycles. Our work can provide a good guidance for the controllable synthesis of other transition metal NASICON-type electrode materials

YTHDF1 Regulates Tumorigenicity and Cancer Stem Cell-Like Activity in Human Colorectal Carcinoma

YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1) is a core factor in RNA methylation modification. Recent studies have shown that m6A is closely related to multiple tumors, thus YTHDF1 may also play a role in tumorigenesis. This study, aimed to explore the role of YTHDF1 in the colorectal cancer (CRC). In this study, we identified YTHDF1 as being highly expressed at the mRNA and protein levels in TCGA, GEO CRC and primary CRC. Furthermore, the YTHDF1 gene copy number was positively correlated with YTHDF1 mRNA expression in CRC. Knocking down the expression of YTHDF1 significantly inhibited the CRC cell's tumorigenicity in vitro and murine xenograft tumor growth in vivo. Furthermore, silencing of YTHDF1 inhibited the colonosphere formation ability in vitro. Mechanistically, we found that silencing YTHDF1 significantly inhibited Wnt/β-catenin pathway activity in CRC cells. Together, YTHDF1 is overexpressed in CRC and plays a vital oncogenic role in CRC, and this novel finding may provide a potential therapeutic target for CRC

Bioresour. Technol.

In this study, pinewood and corn stover pretreated with the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride (AmimCl) were used as a feedstock for succinic acid production. Results reveal that 5% (v/v) AmimCl inhibited bacterial growth, whereas 0.01% (v/v) AmimCl inhibited succinic acid production. AmimCl was effective in extracting cellulose from pinewood and in degrading pinewood into a uniform pulp, as revealed by scanning electron microscopy (SEM). The rate of enzymatic hydrolysis of pinewood extract reached 72.16%. The combinations of AmimCl pretreatment with steam explosion or with hot compressed water were effective in treating corn stover, whereas AmimCl treatment alone did not result in a significant improvement. Pinewood extract produced 20.7 g/L succinic acid with an average yield of 0.37 g per gram of biomass. Workflow calculations indicated pine wood pretreated with IL has a theoretical yield of succinic acid of 57.1%. IL pretreatment led to increase in succinic acid yields. (C) 2014 Elsevier Ltd. All rights reserved.In this study, pinewood and corn stover pretreated with the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride (AmimCl) were used as a feedstock for succinic acid production. Results reveal that 5% (v/v) AmimCl inhibited bacterial growth, whereas 0.01% (v/v) AmimCl inhibited succinic acid production. AmimCl was effective in extracting cellulose from pinewood and in degrading pinewood into a uniform pulp, as revealed by scanning electron microscopy (SEM). The rate of enzymatic hydrolysis of pinewood extract reached 72.16%. The combinations of AmimCl pretreatment with steam explosion or with hot compressed water were effective in treating corn stover, whereas AmimCl treatment alone did not result in a significant improvement. Pinewood extract produced 20.7 g/L succinic acid with an average yield of 0.37 g per gram of biomass. Workflow calculations indicated pine wood pretreated with IL has a theoretical yield of succinic acid of 57.1%. IL pretreatment led to increase in succinic acid yields. (C) 2014 Elsevier Ltd. All rights reserved

A chord error conforming tool path B-spline fitting method for NC machining based on energy minimization and LSPIA

Piecewise linear (G01-based) tool paths generated by CAM systems lack G1 and G2 continuity. The discontinuity causes vibration and unnecessary hesitation during machining. To ensure efficient high-speed machining, a method to improve the continuity of the tool paths is required, such as B-spline fitting that approximates G01 paths with B-spline curves. Conventional B-spline fitting approaches cannot be directly used for tool path B-spline fitting, because they have shortages such as numerical instability, lack of chord error constraint, and lack of assurance of a usable result. Progressive and Iterative Approximation for Least Squares (LSPIA) is an efficient method for data fitting that solves the numerical instability problem. However, it does not consider chord errors and needs more work to ensure ironclad results for commercial applications. In this paper, we use LSPIA method incorporating Energy term (ELSPIA) to avoid the numerical instability, and lower chord errors by using stretching energy term. We implement several algorithm improvements, including (1) an improved technique for initial control point determination over Dominant Point Method, (2) an algorithm that updates foot point parameters as needed, (3) analysis of the degrees of freedom of control points to insert new control points only when needed, (4) chord error refinement using a similar ELSPIA method with the above enhancements. The proposed approach can generate a shape-preserving B-spline curve. Experiments with data analysis and machining tests are presented for verification of quality and efficiency. Comparisons with other known solutions are included to evaluate the worthiness of the proposed solution

Simultaneous Removal of Thiophene and Dibenzothiophene by Immobilized Pseudomonas delafieldii R-8 cells

Biodesulfurization (BDS) is a promising technology for deep desulfurization. In this work, Pseudomonas delafieldii R-8 cells are immobilized in calcium alginate beads and used for BDS of transportation fuels. It is found that thiophene and dibenzothiophene (DBT) can be simultaneously metabolized by immobilized R-8 cells. The initial sulfur content in the model oil is 300 mg·kg ?1 (thiophene: UDBT1:U1). After 10 h of treatment, the thiophene concentration is reduced by 40%, while DBT is reduced by 25%. The utilization rate of thiophene is faster than that of DBT. Moreover, the oi

Bioresour. Technol.

Succinic acid is an important precursor for the synthesis of high-value-added products. Saccharomyces cerevisiae is a suitable platform for succinic acid production because of its high tolerance towards acidity. In this study, a modified pathway for succinate production was established and investigated in S. cerevisiae. The engineered strain could produce up to 6.17 +/- 0.34 g/L of succinate through the constructed pathway. The succinate titer was further improved to 8.09 +/- 0.28 g/L by the deletion of GPD1 and even higher to 9.98 +/- 0.23 g/L with a yield of 0.32 mol/mol glucose through regulation of biotin and urea levels. Under optimal supplemental CO2 conditions in a bioreactor, the engineered strain produced 12.97 +/- 0.42 g/L succinate with a yield of 0.21 mol/mol glucose at pH 3.8. These results demonstrated that the proposed engineering strategy was efficient for succinic acid production at low pH value. (C) 2014 Elsevier Ltd. All rights reserved.Succinic acid is an important precursor for the synthesis of high-value-added products. Saccharomyces cerevisiae is a suitable platform for succinic acid production because of its high tolerance towards acidity. In this study, a modified pathway for succinate production was established and investigated in S. cerevisiae. The engineered strain could produce up to 6.17 +/- 0.34 g/L of succinate through the constructed pathway. The succinate titer was further improved to 8.09 +/- 0.28 g/L by the deletion of GPD1 and even higher to 9.98 +/- 0.23 g/L with a yield of 0.32 mol/mol glucose through regulation of biotin and urea levels. Under optimal supplemental CO2 conditions in a bioreactor, the engineered strain produced 12.97 +/- 0.42 g/L succinate with a yield of 0.21 mol/mol glucose at pH 3.8. These results demonstrated that the proposed engineering strategy was efficient for succinic acid production at low pH value. (C) 2014 Elsevier Ltd. All rights reserved

Simultaneous Removal of Thiophene and Dibenzothiophene by Immobilized Pseudomonas delafieldii R-8 cells

Biodesulfurization (BDS) is a promising technology for deep desulfurization. In this work, PseudomonaS delafieldii R-8 cells are immobilized in calcium alginate beads and used for BDS of transportation fuels. It is found that thiophene and dibenzothiophene (DBT) can be simultaneously metabolized by immobilized R-8 cells. The initial sulfur content in the model oil is 300 mg.kg(-1) (thiophene : DBT = 1 : 1). After 10 h of treatment, the thiophene concentration is reduced by 40%, while DBT is reduced by 25%. The utilization rate of thiophene is faster than that of DBT. Moreover, the oil/water ratio of alginate immobilized cells is studied to reduce the water volume in desulfurization systems. Long-term recycling of BDS by alginate immobilized cells is carried out with oil/water ratio at 5 : 1. The immobilized cells are successfully reused over 15 batch cycles. In the last batch, the desulfurization activity remains at least 75% of the first batch

Clarification of succinic acid fermentation broth by ultrafiltration in succinic acid biorefinery

BACKGROUND: Succinic acid is an important precursor chemical for the synthesis of high value-added products. In this work, ultrafiltration was first investigated to clarify succinic acid fermentation broth by integrating fermentation and separation and removal processes of the product in situ. Four different ultrafiltration membranes (PES 100 kDa, PES 30 kDa, PES 10 kDa and RC 10 kDa) were used in this work. RESULTS: Results indicate that ultrafiltration is feasible for clarifying succinic acid fermentation broth. Almost all the microorganism cells (99.6%) were removed from the fermentation broth. Proteins were also removed effectively by all the membranes studied. The removal rate was 79.86% for PES 100 kDa, 86.43% for PES 30 kDa, 86.83% for PES 10 kDa, and 80.06% for the RC 10 kDa. After ultrafiltration, a clearer permeate was obtained compared with that from centrifugation. CONCLUSION: Membranes operating at high flux are always susceptible to rapid fouling. Compared with molecular weight cut-offs (MWCO), membrane material has a significant influence on the flux. Membrane flux measured in this study shows industrial potential of this technology in treatment of succinic acid fermentation broth. (c) 2012 Society of Chemical Industr

Heterotrophic nitrification and aerobic denitrification by a novel Halomonas campisalis

A novel halophilic strain that could carry out heterotrophic nitrification and aerobic denitrification was isolated and named as Halomonas campisalis ha3. It removed inorganic nitrogen compounds (e.g. NO3 (-), NO2 (-) and NH4 (+)) simultaneously, and grew well in the medium containing up to 20 % (w/v) NaCl. PCR revealed four genes in the genome of ha3 related to aerobic denitrification: napA, nirS, norB and nosZ. The optimal conditions for aerobic denitrification were pH 9.0, at 37 A degrees C, with 4 % (w/v) NaCl and sodium succinate as carbon source. The nitrogen removal rate was 87.5 mg NO3 (-)-N l(-1) h(-1). Therefore, this strain is a potential aerobic denitrifier for the treatment of saline wastewater