Preparation of Dual-Layered Core–Shell Fe3O4@SiO2 Nanoparticles and Their Properties of Plasmid DNA Purification

The rapid purification of biomaterials such as DNA, RNA, and antibodies has attracted extensive attention, and research interest has increased further with the COVID-19 pandemic. In particular, core–shell-structured superparamagnetic nanoparticles have been continuously studied for their application as biopurification materials. It has been reported that Fe3O4@SiO2 nanoparticles are one of the most promising candidates for separating nucleic acids via a simple and rapid process. This study proposed a fabrication method for dual-layered Fe3O4@SiO2 nanoparticles, in which the density of the SiO2 shell was controlled using an intermediate surfactant during the SiO2 coating. After the fabrication of dual-layered Fe3O4@SiO2 nanoparticles, structural, morphological, and magnetic analyses were conducted. The results showed that the Fe3O4 nanoparticles were surrounded by a dense layer 15.6~27.9 nm thick and a porous layer 24.2~44.4 nm thick, and had superparamagnetic properties with high saturated magnetization at room temperature (86.9 emu/g). Then, the optimal conditions for the biopurification material were suggested based on analysis of the selective separation of plasmid DNA.

Instant Taq: Rapid Autoinducible Expression and Chromatography-free Purification of Taq polymerase.

DNA modifying enzymes are ubiquitous reagents in synthetic biology. Producing these enzymes often requires large culture volumes, purified nucleases and chromatographic separations to make enzymes of necessary quality. We sought to leverage synthetic biology tools to develop engineered strains allowing for not only the production but rapid purification of these reagents. Toward this goal, we report an E. coli strain enabling the rapid production and purification of Taq polymerase. The method relies on 1) autoinducible expression achieving high protein titers, 2) autolysis and auto DNA/RNA hydrolysis via lysozyme and a mutant benzonase, and 3) heat denaturation under reducing conditions to precipitate contaminating proteins including the mutant benzonase. Taq polymerase is obtained at high purities (>95% pure by SDS-PAGE) and is readily usable in standard reactions. The method takes less than 1 hour of hands-on time, does not require special equipment, expensive reagents or affinity purification. We expect this simple methodology and approach will improve access not only to Taq polymerase but to numerous additional commonly utilized reagent proteins.

An Efficient and Cost-effective Purification Methodology for Cas9 Nuclease

With an ever-increasing demand for laboratory-grade Cas9 proteins by many groups advancing the use of CRISPR technology, a more efficient and scalable process for generating the proteins, coupled with rapid purification methods is in urgent demand. Here, we introduce a modified methodology for rapid purification of active SaCas9 protein within 24 hours. The product has over 90% protein purity. The simplicity and cost-effectiveness of such methodology will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating the advancement of CRISPR/Cas9-based research.

Rapid purification and metabolomic profiling of synaptic vesicles from mammalian brain

Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of SV content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. In addition, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type-specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.