Programmed gRNA Removal System for CRISPR-Cas9-Mediated Multi-Round Genome Editing in Bacillus subtilis

CRISPR/Cas9 has become a simple and powerful genome editing tool for many organisms. However, multi-round genome editing should replace single-guide RNA (sgRNA) every round, which is laborious and time-consuming. Here, we have developed a multi-round genome editing system in which genome editing and the programmed removal of the sgRNA have sequentially occurred in a growth-dependent manner in Bacillus subtilis. The system contains two plasmids, one containing a cas9 gene and the other containing two sgRNAs and a donor DNA for homology directed repair (HDR). The two sgRNAs are chromosome-targeting (sgRNAct) and self-targeting (sgRNAst) under the control of a constitutive promoter and sporulation-specific promoter, respectively. In the growth phase, the sgRNAct is transcribed and complexed with the Cas9 to edit the chromosomal target, while the sgRNAst is transcribed in the sporulation phase and complexed with the Cas9 to attack its own plasmid. Therefore, the system automatically makes the cell ready for next-round genome editing during cultivation. The system was approved through the sequential deletion of eight extracellular protease genes in the B. subtilis, suggesting that it can be used for versatile applications in multi-round genome editing

Transcriptional Regulation of the rsbV Promoter Controlling Stress Responses to Ethanol, Carbon Limitation, and Phosphorous Limitation in Bacillus subtilis

The σB-dependent promoter in front of the rsbV gene of Bacillus subtilis is induced ∼5-fold in response to (1) the addition of 4% ethanol, (2) carbon starvation, and (3) phosphorous starvation. Binding sites for the global carbon and nitrogen regulators, CcpA and TnrA, were mutated, and the consequences of their loss and that of CcpA or TnrA were studied using rsbV-lacZ fusions. These responses proved to be dependent on CcpA, TnrA, and their putative binding sites upstream of the promoter. Induction in response to glucose limitation was largely abolished by loss of CcpA or the upstream region, while induction in response to phosphorous limitation was largely abolished only by the upstream mutations. The results suggest that CcpA directly influences the carbon starvation response and that both proteins exert indirect effects on all three stress responses. The integrity of the DNA sequence is important for all three responses

Accounting Conservatism, Changes In Real Investment, And Analysts Earnings Forecasts

This study examines whether sell-side analysts fully incorporate into their earnings forecasts the joint effects between accounting conservatism and changes in real investment on the quality of current earnings. Our results indicate that sell-side analysts do not fully incorporate such effects when they forecast future earnings so that they overestimate (underestimate) future earnings when current earnings are inflated (depressed) by those effects. Thus, we conclude that sell-side analysts do not recognize fully the joint effects between accounting conservatism and real activity on the earnings quality and that they need to mitigate their bias to enhance market efficiency by providing investors with a good benchmark for their earnings expectation

A Compact Optical Six-Axis Force/Torque Sensor for Legged Robots Using a Polymorphic Calibration Method

This paper presents a novel design for a compact, lightweight 6-axis force/torque sensor intended for use in legged robots. The design promotes easy manufacturing and cost reduction, while introducing innovative calibration methods that simplify the calibration process and minimize effort. The sensor's advantages are achieved by streamlining the structure for durability, implementing noncontact sensors, and providing a wider sensing range compared to commercial sensors. To maintain a simple structure, the paper proposes a force sensing scheme using photocouplers where the sensing elements are aligned in-plane. This strategy enables all sensing elements to be fabricated on a single printed circuit board, eliminating manual labor tasks such as bonding and coating the sensing elements. The prototype sensor contains only four parts, costs less than $250, and exhibits high response frequency and performance. Traditional calibration methods present challenges, such as the need for specialized equipment and extensive labor. To facilitate easy calibration without the need for specialized equipment, a new method using optimal control is proposed. To verify the feasibility of these ideas, a prototype six-axis F/T sensor was manufactured. Its performance was evaluated and compared to a reference F/T sensor and previous calibration methods.Comment: 12 pages, 13 figures, 9 table

Flow Characteristics Around Step-Up Street Canyons with Various Building Aspect Ratios

We investigate the flow characteristics around step-up street canyons with various building aspect ratios (ratio of along-canyon building length to street-canyon width, and upwind building height to downwind building height) using a computational fluid dynamics (CFD) model. Simulated results are validated against experimental wind-tunnel results, with the CFD simulations conducted under the same building configurations as those in the wind-tunnel experiments. The CFD model reproduces the measured in-canyon vortex, rooftop recirculation zone above the downwind building, and stagnation point position reasonably well. We analyze the flow characteristics, focusing on the structural change of the in-canyon flows and the interaction between the in- and around-canyon flows with the increase of building-length ratio. The in-canyon flows undergo development and mature stages as the building-length ratio increases. In the development stage (i.e., small building-length ratios), the position of the primary vortex wanders, and the incoming flow closely follows both the upstream and downstream building sidewalls. As a result, increasing momentum transfer from the upper layer contributes to a momentum increase in the in-canyon region, and the vorticity in the in-canyon region also increases. In the mature stage (i.e., large building-length ratios), the primary vortex stabilizes in position, and the incoming flow no longer follows the building sidewalls. This causes momentum loss through the street-canyon lateral boundaries. As the building-length ratio increases, momentum transfer from the upper layer slightly decreases, and the reverse flow, updraft, and streamwise flow in the in-canyon region also slightly decrease, resulting in vorticity reduction

Chronicle of a Soil Bacterium: Paenibacillus polymyxa E681 as a Tiny Guardian of Plant and Human Health

The Gram-positive rhizosphere bacterium Paenibacillus polymyxa promotes plant growth and produces various antibiotics. Herein, we review research on this species over the past two and a half decades, and focus on the mechanisms of P. polymyxa strain E681, isolated from barley roots in the South Korea in 1995. Strain E681 has outstanding growth-promoting effects on barley, cucumber, pepper, sesame, and Arabidopsis thaliana and produces antimicrobial compounds that protect plants against pathogenic fungi, oomycetes, and bacteria. Induced systemic resistance elicited by treating seeds or roots with strain E681 is a possible mechanism for protecting systemic plant tissues from biotic and other environmental stresses. Genome sequencing has broadened our horizons for antibiotic development and other industrial applications beyond agricultural use. At least six gene clusters for the biosynthesis of antibiotics have been discovered, including polymyxin (pmx), which was recently re-instated as an antibiotic of last resort against Gram-negative drug-resistant bacteria. Three groups of antibiotic synthetases include the gene clusters that encode one for the non-ribosomal peptide polymyxin, fusaricidin, and tridecaptin, another for the lantibiotic paenilan, and the third for a polyketide. We successfully introduced the pmx gene cluster into the surrogate host Bacillus subtilis and created polymyxin derivatives by domain swapping. Furthermore, various E681 derivatives, including a high fusaricidin producer and strains lacking multi-antibiotics production, have been constructed by random mutagenesis and genome engineering. Thus, E681 is an important bacterium that contributes to both plant and human health

A Highly Efficient CRISPR-Cas9-Mediated Large Genomic Deletion in Bacillus subtilis

In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the eco-friendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis

A Feasibility Study on the Application of TVDI on Accessing Wildfire Danger in the Korean Peninsula

Wildfire is a major natural disaster affecting socioeconomics and ecology. Remote sensing data have been widely used to estimate the wildfire danger with an advantage of higher spatial resolution. Among the several wildfire related indices using remote sensing data, Temperature Vegetation Dryness Index (TVDI) assesses wildfire danger based on both Normalized Difference Vegetation Index (NDVI) and Land Surface Temperature (LST). Although TVDI has physical advantages by considering both weather and vegetation condition, previous studies have shown TVDI does not performed well compare to other wildfire related indices over the Korean Peninsula. In this study we have attempted multiple modification to improve TVDI performance over the study region. In-situ measured air temperature was employed to increase accuracy, regression line was generated using monthly data to include seasonal effect, and TVDI was calculated at each province level to consider vegetation type and local climate. The modified TVDI calculation method was evaluated in wildfire cases and showed significant improvement in wildfire danger estimation

Primary Calcified T-Cell Lymphoma of the Urinary Bladder: A Case Report

Primary malignant lymphoma of the urinary bladder is extremely rare, and to our knowledge, no case described in the radiologic literature has been accompanied by calcification. We report a case in which the condition was associated with calcification, and describe the pelvic CT and MR imaging findings