The trans DNA cleavage activity of Cas12a provides no detectable immunity against plasmid or phage

Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans in vitro. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in Escherichia coli, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity in vivo

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Impact of Water Saving Policy on Water Resource and Economy for Hebei, China Based on an Improved Computable General Equilibrium Model

Hebei Province of China is facing a severe water resource shortage, making it urgent to formulate economical and effective water conservation policies. However, few studies have focused on analyzing the resource and economic impacts of a water policy. This study developed an improved computable general equilibrium (CGE) model with an extended water resources module as a policy analysis tool. The extended water resources module includes different water resources as commodities and water sectors, and introduces a substitution mechanism among the water resources. Policy scenarios containing different policy types and policy objects were established, including water price, technology (tech) improvement, structure adjustment, and water reuse policies in primary, secondary, and tertiary sectors. The impact on the water resource and economy of the scenarios was analyzed using the CGE model. The recommended policies include: an agricultural technology improvement policy that decreases groundwater usage by 240 hm3; an industrial technology improvement policy under which water usage per 10,000 CNY of industrial added value decreases by 13%; an industrial water reuse policy that increases unconventional water usage by 20%; and a structure adjustment policy to increase the proportion of the tertiary sector. The study provides an analysis tool for simulating and evaluating a water resource policy