Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.

BACKGROUND: Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. RESULTS: To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

Application of three-dimensional reconstruction and visible simulation technique in reoperation of hepatolithiasis.

BACKGROUND AND AIM: Hepatolithiasis often requires repeated operations in East Asia. This study aims to evaluate the clinical application of three-dimensional reconstruction and visible simulation techniques for repeated operation in patients with intrahepatic calculi. METHODS: A medical image processing system was used for modeling, segmentation, and three-dimensional reconstruction of intrahepatic stones in 20 patients, consisting of 7 males and 13 females who were subjected to repeated surgical treatment from May 2010 to November 2011. The three-dimensional models of the liver and bile ducts in a standard template library format were then processed by the FreeForm Modeling System. Accurate digital information about the bile duct system, lesions, calculi distribution, and surrounding organs obtained from all directions, multiple angles, and multistrata were used to decide the rational surgical modality. Virtual operations were then performed on the models with virtual surgical instruments in the FreeForm Modeling System. The results were used to guide and were compared with the real surgical procedures performed. RESULTS: The surgical outcomes of all patients in this study were satisfactory. Three-dimensionally reconstructed models provided clear and strong relief perception and a user-friendly interface. Visible simulation surgery performed based on three-dimensionally reconstructed models led to an optimal operation plan that had great resemblance to the actual surgeries for cases with intrahepatic stones. CONCLUSIONS: Three-dimensional reconstruction and visible simulation techniques had unique value in optimizing repeated operation plans and in guiding actual surgical procedures for patients with recurrent intrahepatic calculi.

Lactobacillus rhamnosus and L. plantarum Combination Treatment Ameliorated Colitis Symptoms in a Mouse Model by Altering Intestinal Microbial Composition and Suppressing Inflammatory Response.

SCOPE: Changes in composition of intestinal microbes may disrupt the balance of their interaction with a susceptible host, resulting in development of inflammatory bowel disease (IBD). METHODS AND RESULTS: The study applied in combination two Lactobacillus strains (L. rhamnosus BY-02, L. plantarum BY-05) ("LS treatment"), previously isolates from feces of healthy human infants, in a mouse model of dextran sodium sulfate (DSS)-induced colitis, and evaluates their ameliorative effect and its possible mechanism. LS treatment suppresses weight loss and colon shortening, and reduces disease activity index in the mice. It also has several additional beneficial effects: i) maintains goblet cell numbers and ameliorates intestinal barrier damage in colonic tissue; ii) alters intestinal microbial composition close to normal by increasing abundances of Muribaculaceae, Akkermansia, Clostridia, Oscillospiraceae, and Lachnospiraceae, and decreasing abundance of Escherichia-Shigella; iii) increases content of short-chain fatty acids; iv) reduces content of pro-inflammatory lipopolysaccharides; v) suppresses overactivation of TLR4/NF-κB inflammatory signaling pathway. CONCLUSION: Combination treatment with two Lactobacillus strains strongly ameliorates colitis symptoms in the mouse model by favorably altering intestinal microbial composition and suppressing inflammatory response.

Quantitative Acetylome Analysis of Soft Wheat Seeds during Artificial Ageing.

Lysine acetylation (Kac) is a protein post-translational modification (PTM) widely found in plants that plays vital roles in metabolic pathways. Although seed germination and development are regulated by Kac, its potential function in seed ageing remains to be investigated. Our preliminary study demonstrated that Kac levels were altered during wheat seed artificial ageing. However, its specific role in this process still needs to be elucidated. Here, we performed quantitative acetylation proteomics analysis of soft wheat seeds with different germination rates during artificial ageing. A total of 175 acetylation proteins and 255 acetylation modification sites were remarkably changed. The differentially acetylated proteins were enriched in metabolism; response to harsh intracellular environment, such as ROS; protein storage and processing. Notably, expression, point mutation to mimic Kac by K to Q mutation at K80 and K138, protein purification and enzyme activity detection revealed that the Kac of ROS-scavenging glutathione transferase attenuated its activity, indicating that the defense ability of wheat seeds to stress gradually diminished, and the ageing process was inevitable. Collectively, our data provide a basis for further understanding the roles of Kac in seed ageing and might aid in the development of new techniques to prolong seed viability and food quality.