Reviewer
Lionel Schiavolin
  • Post-Doc, Université Libre de Bruxelles
Research fields
  • Microbiology, Phage biology
Method for Large-scale Production of hIPSC Spheroids
Authors:  Lucas Lemarié, Edwin-Joffrey Courtial and Jérôme Sohier, date: 04/05/2024, view: 681, Q&A: 0

Stem cell spheroids are rapidly becoming essential tools for a diverse array of applications ranging from tissue engineering to 3D cell models and fundamental biology. Given the increasing prominence of biotechnology, there is a pressing need to develop more accessible, efficient, and reproducible methods for producing these models. Various techniques such as hanging drop, rotating wall vessel, magnetic levitation, or microfluidics have been employed to generate spheroids. However, none of these methods facilitate the easy and efficient production of a large number of spheroids using a standard 6-well plate. Here, we present a novel method based on pellet culture (utilizing U-shaped microstructures) using a silicon mold produced through 3D printing, along with a detailed and illustrated manufacturing protocol. This technique enables the rapid production of reproducible and controlled spheroids (for 1 × 106 cells, spheroids = 130 ± 10 μm) from human induced pluripotent stem cells (hIPSCs) within a short time frame (24 h). Importantly, the method allows the production of large quantities (2 × 104 spheroids for 1 × 106 cells) in an accessible and cost-effective manner, thanks to the use of a reusable mold. The protocols outlined herein are easily implementable, and all the necessary files for the method replication are freely available.


Key features

• Provision of 3D mold files (STL) to produce silicone induction device of spheroids using 3D printing.

• Cost-effective, reusable, and autoclavable device capable of generating up to 1.2× 104 spheroids of tunable diameters in a 6-well plate.

• Spheroids induction with multiple hIPSC cell lines.

• Robust and reproducible production method suitable for routine laboratory use.


Graphical overview



Spheroid induction process following the pellet method on molded silicon discs

Rapid Identification of Pathogens in Severe Pneumonia by Species-specific Bacterial Detector (SSBD)
Authors:  Cong Zhang, Xiaohui Liang, Yali Qin, Wenkui Yu and Qihan Chen, date: 05/20/2023, view: 481, Q&A: 0

Fast and accurate detection of pathogenic bacterial infection in patients with severe pneumonia is significant to its treatment. The traditional culture method currently used by most medical institutions relies on a time-consuming culture process (over two days) that is unable to meet clinical needs. Rapid, accurate, and convenient species-specific bacterial detector (SSBD) has been developed to provide timely information on pathogenic bacteria. The SSBD was designed based on the fact that Cas12a indiscriminately cleaves any DNA following the binding of the crRNA-Cas12a complex to the target DNA molecule. SSBD involves two processes, starting with PCR of the target DNA using primers specific for the pathogen, followed by detection of the existence of pathogen target DNA in the PCR product using the corresponding crRNA and Cas12a protein. Compared to the culture test, the SSBD can obtain accurate pathogenic information in only a few hours, dramatically shortening the detection time and allowing more patients to benefit from timely clinical treatment.

Extraction and Electrophoretic Analysis of Bacterial Lipopolysaccharides and Outer Membrane Proteins
Authors:  Yue-Jia Lee and Thomas J. Inzana, date: 12/20/2021, view: 3543, Q&A: 0

Lipopolysaccharides (LPS) (or lipooligosaccharides [LOS], which lack the O-antigen side chains characteristic of LPS), and outer membrane proteins (OMP) are major cell-surface molecules in the outer membrane (OM) of gram-negative bacteria. The LPS is responsible for causing endotoxic shock in infected hosts and, in conjunction with some OMPs, provides protection to the bacterium against host innate immune defenses and attachment to host cells. Electrophoretic analysis can provide valuable information regarding the size, number, and variability of LPS/LOS and OMP components between bacterial strains and mutants, which aids in understanding the basic biology and virulence factors of a particular species. Furthermore, highly purified extracts are normally not required if only electrophoretic analysis is to be done, and various methods have been established for such procedures. Here, we review ameliorated procedures for fast and convenient extraction of LPS/LOS and protein-enriched outer membranes (PEOM) for optimal electrophoretic resolution. Specifically, we will describe the phenol-based micro-method for LPS/LOS extraction, a differential extraction procedure with sodium lauryl sarcosinate for PEOM, and gel preparation for electrophoretic analysis of LPS/LOS samples in detail.



Graphic abstract:



Workflow for the preparation and analysis of LPS/LOS and PEOM.


Preparation and Characterization of Internally Modified DNA Templates for Chemical Transcription Roadblocking
Author:  Eric J. Strobel, date: 09/05/2021, view: 1913, Q&A: 0

Site-specific transcription arrest is the basis of emerging technologies that assess nascent RNA structure and function. Cotranscriptionally folded RNA can be displayed from an arrested RNA polymerase (RNAP) for biochemical manipulations by halting transcription elongation at a defined DNA template position. Most transcription “roadblocking” approaches halt transcription elongation using a protein blockade that is non-covalently attached to the template DNA. I previously developed a strategy for halting Escherichia coli RNAP at a chemical lesion, which expands the repertoire of transcription roadblocking technologies and enables sophisticated manipulations of the arrested elongation complexes. To facilitate this chemical transcription roadblocking approach, I developed a sequence-independent method for preparing internally modified dsDNA using PCR and translesion synthesis. Here, I present a detailed protocol for the preparation and characterization of internally modified dsDNA templates for chemical transcription roadblocking experiments.


Graphic abstract:



Precise transcription roadblocking using functionalized DNA lesions


Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates
Authors:  Joshua E. Mayfield, Seema Irani and Yan Zhang, date: 06/20/2020, view: 3555, Q&A: 0
Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.
Propagation and Purification of Chlamydia trachomatis Serovar L2 Transformants and Mutants
Authors:  Robert Faris and Mary M. Weber, date: 12/20/2019, view: 3682, Q&A: 0
Chlamydia trachomatis (C.t.) is an obligate intracellular pathogen that cannot be cultured axenically and must be propagated within eukaryotic host cells. There are at least 15 distinct chlamydial serovariants that belong to 2 major biovars commonly referred to as trachoma and lymphogranuloma venereum (LGV). The invasive chlamydia LGV serovar L2 is the most widely used experimental model for studying C.t. biology and infection and is the only strain with reliable genetic tools available. New techniques to genetically manipulate C.t. L2 have provided opportunities to make mutants using TargeTron and allelic exchange as well as strains overexpressing epitope-tagged proteins, in turn necessitating the regular purification of transformant and mutant clones. Purification of C.t. is a labor-intensive exercise and one of the most common reagents classically used in the purification process, Renografin, is no longer commercially available. A similar formulation of diatrizoate meglumine called Gastrografin is readily available and we as well as others have had great success using this in place of Renografin for chlamydial purifications. Here, we provide a detailed general protocol for infection, propagation, purification, and titering of Chlamydia trachomatis serovar L2 with additional notes specifically pertaining to mutants or recombinant DNA carrying clones.
Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation
Authors:  Rudolf O Schlechter and Mitja NP Remus-Emsermann, date: 04/05/2019, view: 6905, Q&A: 0
Recently, we published a large and versatile set of plasmids, the chromatic bacteria toolbox, to deliver eight different fluorescent protein genes and four combinations of antibiotic resistance genes to Gram-negative bacteria. Fluorescent tags are important tools for single-cell microbiology, synthetic community studies, biofilm, and host-microbe interaction studies. Using conjugation helper strain E. coli S17-1 as a donor, we show how plasmid conjugation can be used to deliver broad host range plasmids, Tn5 transposons delivery plasmids, and Tn7 transposon delivery plasmids into species belonging to the Proteobacteria. To that end, donor and recipient bacteria are grown under standard growth conditions before they are mixed and incubated under non-selective conditions. Then, transconjugants or exconjugant recipients are selected on selective media. Mutant colonies are screened using a combination of tools to ensure that the desired plasmids or transposons are present and that the colonies are not containing any surviving donors. Through conjugation, a wide range of Gram-negative bacteria can be modified without prior, often time-consuming, establishment of competent cell and electroporation procedures that need to be adjusted for every individual strain. The here presented protocol is not exclusive for the delivery of Chromatic bacteria plasmids and transposons, but can also be used to deliver other mobilizable plasmids to bacterial recipients.
Assessing Yeast Cell Survival Following Hydrogen Peroxide Exposure
Authors:  Khoa Tran and Erin M. Green, date: 01/20/2019, view: 9657, Q&A: 0
In the presence of oxidative stress, cellular defense systems that can detoxify reactive oxygen species are activated through multiple signaling cascades and transcriptional reprogramming. The budding yeast Saccharomyces cerevisiae has served as an excellent model for genetically-identifying factors important for the response to oxidative stress. Here, we describe two assays for testing yeast gene deletion strains or strains overexpressing a gene of interest for viability following oxidative stress induced by hydrogen peroxide treatment. These include a plate-based spot assay for visualizing cell growth and a quantitative colony counting assay. As stress response assays can be highly variable depending on cell growth conditions, these protocols have been optimized for obtaining highly-reproducible results between experiments. We demonstrate the use of these protocols for genetic tests of a putative chromatin regulator implicated in regulating the transcriptional response to oxidative stress.
Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-32P ATP
Authors:  Heather B. Case and Nicholas E. Dickenson, date: 11/05/2018, view: 4352, Q&A: 0
ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase that supports protein secretion through its specialized type three secretion apparatus (T3SA), supporting infection of human host cells. Characterizing ATPases, such as Spa47, requires a means to accurately determine enzyme activity (ATP hydrolysis) as a function of time, reaction conditions, and potential cofactors, regulators, inhibitors, etc. Here, we describe a detailed protocol for characterizing the enzyme kinetics of Spa47 using a direct α-32P ATPase assay.
Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended modification. Here, we describe a CRISPR-Cas9 mediated genome editing protocol for modifying any yeast gene of interest (either essential or nonessential) in a single-step transformation without any selectable marker. In this system, the Cas9 nuclease creates a double-stranded break at the locus of choice, which is typically lethal in yeast cells regardless of the essentiality of the targeted locus due to inefficient non-homologous end-joining repair. This lethality results in efficient repair via homologous recombination using a repair template derived from PCR. In cases involving essential genes, the necessity of editing the genomic lesion with a functional allele serves as an additional layer of selection. As a motivating example, we describe the use of this strategy in the replacement of HEM2, an essential yeast gene, with its corresponding human ortholog ALAD.
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