The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.

Magnetic Biochar Derived from Fenton Sludge/CMC for High-Efficiency Removal of Pb(II): Synthesis, Application, and Mechanism.

Magnetic biochar composites (MBC) were developed by a simple one-step pyrolysis method using Fenton sludge waste solid and carboxymethyl cellulose sodium. Detailed morphological, chemical, and magnetic characterizations corroborate the successful fabrication of MBC. Batch adsorption experiments show that the synthesized MBC owns high-efficiency removal of Pb(II), accompanied by ease-of-separation from aqueous solution using magnetic field. The experiment shows that the equilibrium adsorption capacity of MBC for Pb(II) can reach 199.9 mg g-1, corresponding to a removal rate of 99.9%, and the maximum adsorption capacity (qm) reaches 570.7 mg g-1, which is significantly better than that of the recently reported magnetic similar materials. The adsorption of Pb(II) by MBC complies with the pseudo second-order equation and Langmuir isotherm model, and the adsorption is a spontaneous, endothermic chemical process. Investigations on the adsorption mechanism show that the combination of Pb(II) with the oxygen-containing functional groups (carboxyl, hydroxyl, etc.) on biochar with a higher specific surface area are the decisive factors. The merits of reusing solid waste resource, namely excellent selectivity, easy separation, and simple preparation make the MBC a promising candidate of Pb(II) purifier.

Magnetic supramolecular polymer: Ultrahigh and highly selective Pb(II) capture from aqueous solution and battery wastewater.

For the practical capture of heavy metal ions from wastewater, fabricating environmental friendly adsorbents with high stability and super adsorption capacity are pursuing issue. In this work, we develop magnetic supramolecular polymer composites (M-SMP) by using a simple two-step hydrothermal method. Systematical characterizations of morphological, chemical and magnetic properties were conducted to confirm the formation of M-SMP composites. The resulting M-SMP composites were applied to remove Pb(II) from aqueous solution and from real battery wastewater, and easy separation was achieved using a permanent magnet. By investigating the effects of various parameters, we optimized their operating condition for Pb(II) adsorption by the M-SMP. The uptake of Pb(II) onto M-SMP fitted well the pseudo-second-order and Langmuir isotherm models, and favourable thermodynamics showed a spontaneous endothermic process. The SMP endowed M-SMP with ultrahigh adsorption capacity for Pb(II) (946.9 mg g-1 at pH = 4.0, T = 298 K), remarkable selectivity, satisfactory stability and desirable recyclability. In Pb-contaminated lead-acid battery industrial wastewater, the concentration of Pb(II) declined from 18.070 mg L-1 to 0.091 mg L-1, which meets the current emission standard for the battery industry. These merits, combined with simple synthesis and convenient separation, make M-SMP an outstanding scavenger for the elimination of industrial Pb(II) wastewater.