Search

We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, "tCRISPRi", for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

Abstract Based on a combination of high resolution 3-D seismic, drilling and well logging and core data, this study focuses on describing the depositional features of the Neogene Minghuazhen Formation shallow water delta in Huanghekou area (HHKA), Bohai offshore basin and discussing the evolution and controlling factors of shallow water delta sandbody. An obvious meandering fluvial delta system developed in sequence 1 (SQ1) of the Neogene in HHKA with thinner sandbody of distributary channels and poor development of mouth bar. The sequence texture obviously influences the vertical development and stacking pattern of sandbodies and controls the distribution of sandbodies in plain view as well. In shallow water lacustrine basins, relative topographic height difference leads to change of distribution of accommodation space, and sandbodies of distributary channels usually develop well in local low-lying areas where accommodation space increases. The delta is dominated by distributary channel sandbodies during the early period of base level rising. Sandbodies contact with each other in a lateral stacking pattern and are characterized by a fan shape in plain view. Distributary channels gradually narrow and tend to shift during the mid- late-period of base level rising, while the sandbodies are characterized by a net shape in plain view. During the period of base level slow falling, the multistory/multilateral channel sandbodies dominated the inner front of shallow-water delta and the delta sand dispersal distributes as a lobe shape.

Waterlogging, an abiotic stress, severely restricts crop yield in various parts of the world. Thus, we conducted a meta-analysis of 2,419 comparisons from 115 studies to comprehensively evaluate the overall change in crop yield induced by waterlogging in the global region. The results suggested that waterlogging obviously decreased crop yield by 32.9% on average, compared with no waterlogging, which was a result of a reduced 1,000-grain weight (13.67%), biomass (28.89%), plant height (10.68%), net photosynthetic rate (Pn, 39.04%), and leaf area index (LAI, 22.89%). The overall effect of a waterlogging regime on crop yield is related to the crop type; the crop yield reduction varied between wheat (25.53%) and cotton (59.95%), with an overall average value of 36.81% under field conditions. In addition, we also found that compared with no waterlogging, waterlogging in the reproductive growth stage (41.90%) caused a greater yield reduction than in the vegetative growth stage (34.75%). Furthermore, decreases in crop yield were observed with an extension in the waterlogging duration; the greatest decreases in crop yield occurred at 15 < D ≤ 28 (53.19 and 55.96%) under field and potted conditions, respectively. Overall, the results of this meta-analysis showed that waterlogging can decrease crop yield and was mainly affected by crop type, growth stage, and experimental duration.

Maize and soybean intercropping is a cereal-legume intercropping pattern that not only increases grain yield but also improves the nutritional value of silage. Experiments were conducted in the summer season to compare the yield and nutritional composition of the forage and silage quality of mono-cropped maize and intercropped maize-soybean harvested at two stages of maturity. The main treatments were one sole crop maize (SM) and four maize-soybean intercropping patterns (one-row maize to one-row soybean (1M1S), one-row maize to two-row soybean (1M2S), one-row maize to three-row soybean (1M3S), and two-row maize to one-row soybean (2M1S). The crops were harvested when the maize reached the milk (R3) and maturity (R6) stages. Results indicated a significant increase in the fresh biomass and dry-matter production of maize fodder alone compared with those of maize intercropped with soybean fodder. After 60 days of ensiling period, silage samples were analyzed for pH, organic acids, dry matter, crude protein (CP), ether extract, neutral detergent fiber (NDF), acid detergent fiber (ADF), and other mineral compositions. All intercropped silages showed higher CP values (1M1S, 12.1%; 1M2S, 12.2%; 1M3S, 12.4%; and 2M1S, 12.1%) than the SM silage (8.7%). Higher organic acids were produced in 1M3S than in the other silages. Correlation data showed that CP was highly correlated with lactic acid but negatively associated with crude fiber, nitrogen-free extract, and NDF. Thus, the intercropping of maize and soybean silage is recommended due its enhanced crop production, nutritional values in dairy animals, and prolific animal feedings and because it was scientifically evaluated as a feed stuff. This study indicated that 1M3S was the most preferable among intercropped silages in terms of nutrient composition.

With the help of the variable substitution and applying the fixed point theorem, we derive the sufficient conditions which guarantee the existence of the positive almost periodic solutions for a class of Lotka-Volterra type system. The main results improve and generalize the former corresponding results. [ABSTRACT FROM AUTHOR]

The time estimate of the blow-up of the weakly damped forced KdV equation in thin 2D domains is given. [ABSTRACT FROM AUTHOR]