Protocols in Current Issue
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0 Q&A 173 Views Sep 5, 2023

In the field of molecular genetics, DNA extraction protocols and kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Expanding upon previous fast extraction protocols such as alkaline- and detergent-based ones, the use of boiling-hot water to rupture cells, virions, and nuclei, as proposed during the COVID-19 pandemic, might alleviate shortages and costs. Different soft, relatively abundant (highly enriched), and uncomplicated (genomically homogenous and with few inhibitors) biosamples are collected in 1.5 mL tubes, mixed with boiling-hot water, and stirred vigorously, so as to have membranes lysed and proteins deactivated; mechanical disruption may be used as well if necessary. Incubation in boiling water bath for 20–30 min follows. Depending on sample type and quantity, which affects the total extraction volume, 2–5 μL are pipetted off for direct PCR and the same volume for two decimal serial dilutions. The latter are intended to optimize the crude extract to a workable DNA/inhibitor concentration balance for direct PCR. Uncomplicated, highly enriched samples such as mycelial growth in fruits and human swab samples can be processed, contrary to complicated samples such as blood and physically unyielding samples such as plant tissue. The extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing nucleic acid amplification tests (NAAT) being performed in resource-limited settings with low cost and waste footprint or during prolonged crises, where supply chain failures may occur.

Key features

• DNA extraction from different sample types using only boiling water and occasional mechanical assistance.

• Crude extract serially diluted twice, 10- and 100-fold, to bypass purification and quantification steps.

• Direct PCR for 2–10 μL of crude lysate and dilutions (conditional to sample type and quantity) to enhance probability of workable DNA-inhibitors’ concentrations.

• Lowers the cost and curtails the overall footprint of testing to increase sustainability in field operations and in standard lab environments under supply chain derailment.

0 Q&A 137 Views Sep 5, 2023

Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial–drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs.

Key features

• Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant.

• Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine.

• A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate.

• The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.

Graphical overview

Protocol overview. (1) Bacteria are incubated with gemcitabine for a set period of time. (2) Samples are removed from co-incubated suspensions and filtered to remove bacteria to halt gemcitabine degradation. (3) A gemcitabine-sensitive reporter strain is then added to the conditioned supernatant and is supplemented with growth media. (4) Growth of the reporter strain is monitored over time. (5) Results from the growth experiments are used to infer the concentration of gemcitabine in the co-culture supernatant and the drug degradation rate.

0 Q&A 147 Views Sep 5, 2023

Magnaporthe oryzae is a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain.

Key features

• Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Graphical overview

0 Q&A 786 Views Aug 20, 2023

Lipids can play diverse roles in metabolism, signaling, transport across membranes, regulating body temperature, and inflammation. Some viruses have evolved to exploit lipids in human cells to promote viral entry, fusion, replication, assembly, and energy production through fatty acid beta-oxidation. Hence, studying the virus–lipid interactions provides an opportunity to understand the biological processes involved in the viral life cycle, which can facilitate the development of antivirals. Due to the diversity and complexity of lipids, the assessment of lipid utilization in infected host cells can be challenging. However, the development of mass spectrometry, bioenergetics profiling, and bioinformatics has significantly advanced our knowledge on the study of lipidomics. Herein, we describe the detailed methods for lipid extraction, mass spectrometry, and assessment of fatty acid oxidation on cellular bioenergetics, as well as the bioinformatics approaches for detailed lipid analysis and utilization in host cells. These methods were employed for the investigation of lipid alterations in TMEM41B- and VMP1-deficient cells, where we previously found global dysregulations of the lipidome in these cells. Furthermore, we developed a web app to plot clustermaps or heatmaps for mass spectrometry data that is open source and can be hosted locally or at https://kuanrongchan-lipid-metabolite-analysis-app-k4im47.streamlit.app/. This protocol provides an efficient step-by-step methodology to assess lipid composition and usage in host cells.

Graphical overview

0 Q&A 182 Views Aug 20, 2023

Various photoautotrophic cyanobacteria accumulate intracellular poly(3-hydroxybutyrate) (PHB) granules. This protocol can be used for determining the PHB contents of the cells as % PHB weight per dry cell weight using acid hydrolysis followed by high-performance liquid chromatography (HPLC). This HPLC analysis is rapid, with a running time of approximately 5 min per sample. The technique can accurately determine PHB concentrations in the range of 2–1,000 μg/mL PHB. However, this technique is not applicable for determining the contents of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in cyanobacteria.

0 Q&A 195 Views Aug 5, 2023

The development of antimicrobial resistance and the formation of Salmonella biofilms are serious public health problems. For this reason, new natural compounds with antimicrobial and anti-biofilm activity are being sought, and wild fungi represent an untapped potential. Various extraction agents, including organic solvents and aqueous buffers, can be used to obtain bioactive compounds from natural sources. To evaluate their bioactivity, extensive screening studies are required to determine antimicrobial and anti-biofilm activity using methods such as broth microdilution or crystal violet assay, respectively, but none of these methods allow simultaneous evaluation of both activities against bacteria. Cold water extraction from wild fungi offers the advantage of extracting water-soluble compounds. The SIMultaneous detection of antiMicrobial and anti-Biofilm Activity (SIMBA) method combines the testing of both types of activity against bacteria with the evaluation of the 20 h growth curve of the Salmonella Infantis ŽM9 strain determined with absorbance measurements at 600 nm in a 96-well plate. SIMBA method thus shortens the time to determine the bioactivity of extracts, reduces material consumption, and eliminates the need for additional reagents. SIMBA enables rapid selection of bioactive extracts for their fractionation and shortens the time to determine new natural products with antimicrobial and anti-biofilm activity.

Graphical overview

0 Q&A 226 Views Aug 5, 2023

High yield of good quality plasmid DNA from gram -ve bacteria (Agrobacterium tumefaciens, A. rhizogenes, and Rhizobium sp.) and gram +ve bacterium (Bacillus thuringiensis) is difficult. The widely used plasmid extraction kits for Escherichia coli yield a low quantity of poor-quality plasmid DNA from these species. We have optimized an in-house modification of the QIAprep Spin Miniprep kit protocol of Qiagen, consisting of two extraction steps. In the first, the centrifugation after adding neutralization buffer is followed by ethanol (absolute) precipitation of plasmid DNA. In the second extraction step, the precipitated DNA is dissolved in Tris-EDTA (TE) buffer, followed by an addition of 0.5 volumes of 5 M sodium chloride and 0.1 volumes of 20% (w/v) sodium dodecyl sulfate. After incubation at 65 °C for 15 min, the plasmid DNA is extracted with an equal volume of chloroform:isoamyl alcohol (CIA). RNase (20 mg/mL) is added to the upper phase retrieved after centrifugation and is incubated at 37 °C for 15 min. The extraction of the plasmid DNA with an equal volume of CIA is followed by centrifugation and is precipitated from the retrieved upper phase by adding an equal volume of absolute ethanol. The pellet obtained after centrifugation is washed twice with 70% (v/v) ethanol, air dried, dissolved in TE buffer, and quantified. This easy-to-perform protocol is free from phenol extraction, density gradient steps, and DNA binding columns, and yields high-quality plasmid DNA. The protocol opens an easy scale up to yield a large amount of high-quality plasmid DNA, useful for high-throughput downstream applications.

Key features

• The protocol is free from density gradient steps and use of phenol.

• The protocol is an extension of the QIAprep Spin Miniprep kit (Qiagen) and is applicable for plasmid DNA isolation from difficult-to-extract bacterial species.

• The protocol facilitates the direct transformation of the ligation product into Agrobacterium by skipping the step of E. coli transformation.

• The plasmids isolated are of sequencing grade and the method is useful for extracting plasmids for metagenomic studies.

Graphical overview

Overview of the plasmid isolation protocol (modified QIAprep Spin Miniprep kit) of the present study

0 Q&A 184 Views Aug 5, 2023

Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens–mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering A. tumefaciens to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer A. tumefaciens to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered A. tumefaciens.

0 Q&A 486 Views Jul 20, 2023

Hepatitis B virus (HBV) infection is a global public health concern. During chronic infection, the HBV small-surface antigen is expressed in large excess as non-infectious spherical subviral particles (SVPs), which possess strong immunogenicity. To date, attempts at understanding the structure of HBV spherical SVP have been restricted to 12–30 Å with contradictory conclusions regarding its architecture. We have used cryo-electron microscopy (cryo-EM) and 3D image reconstruction to solve the HBV spherical SVP to 6.3 Å. Here, we present an extended protocol on combining AlphaFold2 prediction with a moderate-resolution cryo-EM density map to build a reliable 3D model. This protocol utilizes multiple software packages that are routinely used in the cryo-EM community. The workflow includes 3D model prediction, model evaluation, rigid-body fitting, flexible fitting, real-space refinement, model validation, and model adjustment. Finally, the described protocol can also be applied to high-resolution cryo-EM datasets (2–4 Å).

0 Q&A 190 Views Jul 20, 2023

An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line–derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture–generated HBV (HBVcc) by the transient transfection of the HBV molecular clone, a large amount of purified viruses is needed, because such viruses exhibit limited infection efficiencies in cell culture. Here, we describe how to generate and purify HBVcc by the transient transfection of HBV molecular clones. This system provides a powerful tool for studying the infection and propagation of HBV and for developing anti-viral agents against HBV.

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