The work was supported by the National Natural Science Foundation of China 82374134.

The experiments conducted adhered to local, national, and international biosafety containment regulations appropriate to the specific biosafety hazards associated with each strain. Fecal samples were collected from healthy volunteers who had not taken any drugs for at least one week. Details of the reagents and equipment used are provided in the Table of Materials.
1. Construction of human intestinal bacterial transformation model&#160;&#160;in vitro
<ol>
	<li>Preparation of General Anaerobic Medium (GAM)25
	<ol>
		<li>Preparation of Solution A
		<ol>
			<li>Combine 10.0 g of tryptone, 10.0 g of proteose peptone, 3.0 g of soya peptone, 13.5 g of digestive serum powder, 1.2 g of liver extract powder, 2.2 g of beef extract, and 5.0 g of yeast extract in a 500 mL Erlenmeyer flask.</li>
			<li>Add 500 mL of distilled water and stir under heating until the substances are dissolved completely.</li>
		</ol>
		</li>
		<li>Preparation of Solution B
		<ol>
			<li>Prepare Solution B following the same procedure as Solution A, except combine 3.0 g of glucose, 5.0 g of soluble starch, 2.5 g of dipotassium hydrogen phosphate, and 3.0 g of sodium chloride in a 250 mL Erlenmeyer flask.</li>
		</ol>
		</li>
		<li>Preparation of Solution C
		<ol>
			<li>Prepare Solution C following the same procedure as Solutions A and B, except combine 0.3 g of L-cysteine hydrochloride and 0.3 g of sodium thioglycolate in a 100 mL Erlenmeyer flask.</li>
			<li>Mix all three solutions in a 1000 mL Erlenmeyer flask, adjust the pH to 7.2 using a 10% sodium hydroxide solution, and add distilled water to make up a final volume of 1000 mL.</li>
			<li>Distribute the medium into 250 mL Erlenmeyer flasks and autoclave at 121 &#730;C for 30 min. Cool to room temperature and store at 4 &#730;C.</li>
		</ol>
		</li>
		<li>Preparation of solid general anaerobic medium
		<ol>
			<li>Add 1%-2% agar to the GAM liquid medium and heat until completely dissolved. Distribute into 250 mL Erlenmeyer flasks and autoclave at 121 &#730;C for 30 min.</li>
		</ol>
		</li>
		<li>Preparation of low-carbon source medium
		<ol>
			<li>Prepare the low-carbon source medium using the same procedure as GAM, but omit yeast extract, glucose, and soluble starch.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of Mixed Human Intestinal Flora
	<ol>
		<li>Collect 1-2 g of fresh human feces in a disposable sterile stool collection tube filled with nitrogen gas. Seal the tube promptly. Add 5 mL of GAM medium, seal, and culture at 37 &#730;C for 24 h in a sterile anaerobic incubator. 
		NOTE: Use an oxygen meter to monitor the anaerobic incubator&#39;s oxygen levels, and replace nitrogen gas as necessary to maintain anaerobic conditions.</li>
	</ol>
	</li>
	<li>Activation of mixed human intestinal flora
	<ol>
		<li>After 24 h, transfer 0.5 mL of the human intestinal bacterial suspension to a new microcentrifuge tube containing 4.5 mL of fresh GAM medium.</li>
		<li>Seal and incubate at 37 &#730;C for 24 h under anaerobic conditions to obtain active mixed human intestinal flora.</li>
	</ol>
	</li>
	<li>Preparation of reference solutions
	<ol>
		<li>Weigh 5 mg each of orientin and luteolin, and dissolve them separately in 1 mL of DMSO to prepare orientin and luteolin reference solutions at a concentration of 5 mg/mL.</li>
	</ol>
	</li>
	<li>Screening of active intestinal flora capable of cleaving C-glycosides
	<ol>
		<li>Mix 260 &#181;L of activated intestinal flora with 6 mL of fresh low-carbon source medium containing 40 &#181;L of orientin reference solution, and incubate at 37 &#730;C.</li>
		<li>Include a control group (6 mL of low-carbon source medium, 40 &#181;L of orientin reference solution, and 260 &#181;L of normal saline) and a blank group (6 mL of low-carbon source medium, 40 &#181;L of normal saline, and 260 &#181;L of activated intestinal flora). 
		NOTE: Perform all tests in triplicate in an anaerobic incubator.</li>
	</ol>
	</li>
	<li>Pretreatment of transformation solutions
	<ol>
		<li>After 24 h and 48 h, pipette 1 mL of the reaction solutions into 1.5 mL microcentrifuge tubes and centrifuge at 13,400 x g for 15 min at room temperature to remove bacteria.</li>
		<li>Add 200 &#181;L of the supernatant to 600 &#181;L of methanol, mix, and centrifuge at 13,400 x g for 15 min at room temperature to remove proteins.</li>
	</ol>
	</li>
	<li>Detection of transformation products
	<ol>
		<li>Filter the supernatant using a 0.22 &#181;m microporous membrane and analyze the filtrate via high performance liquid chromatography (HPLC). Conduct HPLC analysis on an instrument equipped with a C18 column (4.6 mm &#215; 250 mm, 5 &#181;m).</li>
		<li>Maintain the column oven temperature at 40 &#730;C, with a flow rate of 1 mL/min. Use acetonitrile as mobile phase A and 0.1% formic acid in purified water as mobile phase B. 
		NOTE: Employ a linear gradient system as follows: 5%-55% A at 0-20 min, 55%-95% A at 20-25 min, 95%-5% A at 25-30 min, and 5% A at 30-32 min.</li>
	</ol>
	</li>
</ol>
2. Isolation of single strains from human intestinal bacterial flora
<ol>
	<li>Activation of mixed human intestinal bacterial flora
	<ol>
		<li>Activate the mixed human intestinal bacterial flora capable of cleaving orientin, as described in step 1.3, to obtain the activated human intestinal bacterial flora solution.</li>
	</ol>
	</li>
	<li>Preparation of solid GAM plates
	<ol>
		<li>Dissolve the prepared solid GAM medium by heating, and pour it into disposable sterile petri dishes in an anaerobic incubator. Allow the medium to solidify to prepare solid GAM plates.</li>
	</ol>
	</li>
	<li>Preliminary screening of single strains from human intestinal bacterial flora
	<ol>
		<li>Pipette 100 &#181;L of the activated bacterial solution into a 1.5 mL microcentrifuge tube containing 1 mL of normal saline.</li>
		<li>Inoculate an appropriate volume of the bacterial solution onto a petri dish using a disposable inoculation ring. Seal the dish and incubate it in an anaerobic incubator.</li>
	</ol>
	</li>
	<li>Observation of colony morphology
	<ol>
		<li>After 48 h of incubation at 37 &#730;C, observe the size, morphology, and color of colonies on the plate.</li>
	</ol>
	</li>
	<li>Selection of individual colonies to obtain single strains
	<ol>
		<li>Select distinct single bacterial colonies and culture them in 10 mL microcentrifuge tubes containing 4.5 mL of fresh GAM medium. Incubate under anaerobic conditions at 37 &#730;C for 24 h to obtain single strains.</li>
	</ol>
	</li>
	<li>Verification of single strain activity
	<ol>
		<li>Verify the activity of single strains as described in steps 1.5, 1.6, and 1.7. Perform activity verification using high performance liquid chromatography (HPLC) and plate marking25.</li>
	</ol>
	</li>
	<li>Purification of single strains from human intestinal bacterial flora
	<ol>
		<li>Purify the active single strain as described in step 2.1. Verify its activity through transformation experiments with orientin, following the procedures outlined in steps 1.5, 1.6, and 1.7.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

Standard operating procedure&#160;(SOP) for screening human intestinal bacteria capable of cleaving C-glycosides were established. Using these procedures, a pure active strain was successfully obtained, and its deglycosylation property was confirmed through transformation tests. The SOP consists of the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium.
The most important aspect of the screening process is the use of low-carbon source medium to induce bacterial activity and increase the success rate of screening. During transformation experiments, low-activity bacteria are difficult to identify using GAM medium but are more readily detected with low-carbon source medium25. Preparation and enrichment techniques maximize target strains while minimizing non-target strains. The screening process is conducted step by step under the guidance of activity tests, increasing the success rate and minimizing the false positive rate. This SOP can be employed or referenced by researchers seeking to obtain functional bacteria.
In comparison to alternative methodologies, this SOP is specifically designed for screening intestinal bacteria capable of cleaving C-glycosides1,9,18. Challenges remain, including the suitability of low-carbon source medium for screening bacteria with other functional properties and the potential for active strains to be lost during the screening process. If such strains are mistracked, they can be retrieved by repeating the preceding steps25.
In conclusion, an SOP for screening intestinal bacteria capable of cleaving C-glycosides was developed. The target strains can be efficiently screened using this SOP, and the methodology can serve as a reference for researchers aiming to isolate other functional bacteria.

C-glycosides are a group of compounds characterized by the direct linkage of glycosyl groups to aglycones through C-C bonds1. In nature, orientin, vitexin, puerarin, and their derivatives are commonly identified as C-glycosides2. These compounds are frequently found in medicinal plants such as Trollius chinensis3 and in animals such as Styela plicata4. Studies have demonstrated that these compounds provide health benefits and exhibit various bioactivities, including antibacterial5,6,7, anti-inflammatory8,9,10,11,12, antiviral3,12,13, antioxidant14,15,16, and antineoplastic activities17.
Due to the linkage of glycosyl groups to the skeleton through C-C bonds, these compounds exhibit high stability and resistance to acidic and enzymatic hydrolysis18, leading to poor absorbability and low bioavailability19. This limitation affects the development and utilization of C-glycosides. However, drugs containing C-glycosides, often administered orally, are deglycosylated by specific enzymes, producing more potent bioactive aglycones20,21,22,23. While host&#39;s own enzymes are unable to deglycosylate C-glycosides, intestinal bacteria have been reported to metabolize certain types. For instance, some intestinal bacteria convert mangiferin into norathyriol, which exhibits greater potency as an antidiabetic or antineoplastic agent24. Despite these findings, only a limited number of bacteria capable of cleaving C-glycosides have been characterized, and the underlying mechanisms remain poorly understood. Efficient and standardized screening of these bacteria will enhance understanding of their functions and accelerate the development of C-glycosides.
The screening method has been developed and refined over time into a mature system. The core approaches include enrichment, activity-oriented screening, and activity validation in low-carbon source media. This method facilitates the isolation of pure target strains, as well as the identification of species, genomic data, and traits of target microorganisms. Using this system, intestinal bacteria capable of cleaving C-glycosides can be effectively screened and isolated.

<table><tbody><tr><td>Acetonitrile</td><td>Thermo Fisher Scientific</td><td>F2408R205</td><td>HPLC</td></tr><tr><td>Anaerobic Incubator</td><td>Shanghai CIMO Medical Instrument Manufacturing Co., LTD</td><td></td><td>YQX- II</td></tr><tr><td>Beef Extract</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-009</td><td>BR</td></tr><tr><td>Digestive Serum Powder</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-087</td><td>BR</td></tr><tr><td>Dipotassium Hydrogen Phosphate</td><td>Beijing Chemical Works</td><td>M26298</td><td>AR</td></tr><tr><td>Disposable Sterile Stool Collection Tube</td><td>Lang Fu Co., LTD</td><td></td><td>5 mL</td></tr><tr><td>Distilled Water</td><td>Department of Biopharmaceutical, Beijing University of Traditional Chinese Medicine</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich Corporation</td><td>WXBD2861V</td><td>AR</td></tr><tr><td>EP Tube</td><td>Beijing Biodee Biotechnology Co., LTD</td><td></td><td>10 mL/1.5 mL</td></tr><tr><td>Eppendorf Centrifuge</td><td>Eppendorf AG</td><td></td><td>5418</td></tr><tr><td>Glucose</td><td>Beijing Chemical Works</td><td>GC205003</td><td>AR</td></tr><tr><td>High Performance Liquid Chromatograph</td><td>Shimadzu Corporation</td><td></td><td>LC-20</td></tr><tr><td>High-pressure Steam Sterilizer</td><td>Sanyo Denki Shanghai Co., LTD</td><td></td><td>MLS-3780</td></tr><tr><td>Innoval C&lt;sub&gt;18 &lt;/sub&gt;Chromatographic Column</td><td>Agela Technologies Co., LTD</td><td></td><td>4.6 mm &amp;times; 250 mm, 5 &amp;micro;m</td></tr><tr><td>L-cysteine Hydrochloride</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>BGASY01</td><td>BR</td></tr><tr><td>Liver Extract Powder</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-085</td><td>BR</td></tr><tr><td>Luteolin</td><td>National Institutes for Food and Drug Control</td><td></td><td>&gt;98%</td></tr><tr><td>Magnetic Stirrer</td><td>Ika Werke Co., LTD</td><td></td><td>RCT basic</td></tr><tr><td>Methanol</td><td>Thermo Fisher Scientific</td><td>20240901312</td><td>AR</td></tr><tr><td>Millipore Filter Membrane</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td></td><td>0.22 &amp;micro;L &amp;times; 50 mm</td></tr><tr><td>Orientin</td><td>Yishiming (Beijing) Biotechnology Co., LTD</td><td>19120601</td><td>&gt;98%</td></tr><tr><td>Peptone</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>1685787</td><td>BR</td></tr><tr><td>Petri Dish</td><td>Beijing Biodee Biotechnology Co., LTD</td><td></td><td>150 mm</td></tr><tr><td>Sodium Chloride</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>BN20008</td><td>AR</td></tr><tr><td>Sodium Thioglycolate</td><td>Shanghai Jianglai Biotechnology Co., LTD</td><td>J031S219019</td><td>AR</td></tr><tr><td>Soluble Starch</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>S9765</td><td>BR</td></tr><tr><td>Soya Peptone</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>2147955</td><td>BR</td></tr><tr><td>Tryptone</td><td>Agela Technologies Co., LTD</td><td>1685787</td><td>BR</td></tr><tr><td>Ultrasonic Cleaner</td><td>Kun Shan Ultrasonic Instruments Co., LTD</td><td></td><td>KQ-500DE</td></tr><tr><td>Yeast Extract</td><td>Beijing Abxing Biotechnology Co., LTD</td><td>01-014</td><td>BR</td></tr></tbody></table>

screening and isolation of c-glycoside-cleaving intestinal bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

Fecal samples from ten healthy volunteers were screened using transformation experiments, resulting in one sample demonstrating activity in deglycosylating orientin. This finding confirmed the feasibility of screening for active samples. The active sample was isolated using the plate marking method. Based on the morphology and characteristics of the colonies, approximately 18 single bacterial colonies with yellow or white coloration, varied shapes, and uneven edges were selected for further analysis. Among these, only one colony demonstrated the ability to cleave orientin into its aglycone, luteolin. The colony was further purified through five iterations of the plate marking process, following the previously described methodology, to obtain a pure active strain. High performance liquid chromatography (HPLC) analysis was employed to detect deglycosylation products. The retention times for orientin and luteolin were 11.5 min and 16.5 min, respectively (Figure 1). The pure strain with deglycosylation activity was successfully isolated, and its morphological characteristics were documented (Figure 2). On GAM solid medium, the colonies displayed beige coloration with irregular edges.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67346/67346fig01.jpg" /> 
Figure 1: HPLC chromatograms related to deglycosylation of orientin. (A) Reference compounds of orientin and luteolin. (B) Blank group. (C) Experimental group at 24 h. (D) Experimental group at 48 h. a: orientin; b: luteolin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67346/67346fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67346/67346fig02.jpg" /> 
Figure 2: Morphological characteristics of strain. The size of the Petri dish used is 90 mm &#215; 16 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67346/67346fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This study establishes a set of standard operating procedures (SOPs) for efficiently screening and isolating intestinal bacteria capable of cleaving C-glycosides.

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

screening-and-isolation-of-c-glycoside-cleaving-intestinal-bacteria

Nanyang Technological University

Research

JoVE Journal

Biochemistry

235 Views.  Beijing University of Chinese Medicine. Deze studie stelt een reeks standaard operationele procedures (SOP's) vast voor het efficiënt screenen en isoleren van darmbacteriën die in staat zijn om C-glycosiden te splitsen.

Video: Screening en isolatie van C-glycoside-splitsende darmbacteriën

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

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