Test Tube Reactions Research Articles

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library.

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5' end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.

Immobilised-enzyme microreactors for the identification and synthesis of conjugated drug metabolites.

The study of naturally circulating drug metabolites has been a focus of interest, since these metabolites may have different therapeutic and toxicological effects compared to the parent drug. The synthesis of metabolites outside of the human body is vital in order to conduct studies into the pharmacological activities of drugs and bioactive compounds. Current synthesis methods require significant purification and separation efforts or do not provide sufficient quantities for use in pharmacology experiments. Thus, there is a need for simple methods yielding high conversions whilst bypassing the requirement for a separation. Here we have developed and optimised flow chemistry methods in glass microfluidic reactors utilising surface-immobilised enzymes for sulfonation (SULT1a1) and glucuronidation (UGT1a1). Conversion occurs in flow, the precursor and co-factor are pumped through the device, react with the immobilised enzymes and the product is then simply collected at the outlet with no separation from a complex biological matrix required. Conversion only occurred when both the correct co-factor and enzyme were present within the microfluidic system. Yields of 0.97 ± 0.26 μg were obtained from the conversion of resorufin into resorufin sulfate over 2 h with the SULT1a1 enzyme and 0.47 μg of resorufin glucuronide over 4 h for UGT1a1. This was demonstrated to be significantly more than static test tube reactions at 0.22 μg (SULT1a1) and 0.19 μg (UGT1a1) over 4 h. With scaling out and parallelising, useable quantities of hundreds of micrograms for use in pharmacology studies can be synthesised simply.

The 3,4-Quinones of Estrone and Estradiol Are the Initiators of Cancer whereas Resveratrol and N-acetylcysteine Are the Preventers.

This article reviews evidence suggesting that a common mechanism of initiation leads to the development of many prevalent types of cancer. Endogenous estrogens, in the form of catechol estrogen-3,4-quinones, play a central role in this pathway of cancer initiation. The catechol estrogen-3,4-quinones react with specific purine bases in DNA to form depurinating estrogen-DNA adducts that generate apurinic sites. The apurinic sites can then lead to cancer-causing mutations. The process of cancer initiation has been demonstrated using results from test tube reactions, cultured mammalian cells, and human subjects. Increased amounts of estrogen-DNA adducts are found not only in people with several different types of cancer but also in women at high risk for breast cancer, indicating that the formation of adducts is on the pathway to cancer initiation. Two compounds, resveratrol, and N-acetylcysteine, are particularly good at preventing the formation of estrogen-DNA adducts in humans and are, thus, potential cancer-prevention compounds.

CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.

Construction of a genome-wide transgenic UAS-cDNA/ORF library in Drosophila based on the binary GAL4/UAS system has been severely hampered by technical difficulties, although genome-wide cDNA or ORF resources of Drosophila, human, and mouse have been publicly available for more than a decade. Here, we developed a new method named CRISPR-based modular assembly (CRISPRmass) for the high-throughput construction of a genome-wide UAS-cDNA/ORF library from publicly available cDNA/ORF resources. Through cleavage of shared vector sequences of cDNA/ORF plasmids by CRISPR/Cas9 and subsequent insertion of UAS modules by Gibson assembly, the procedure of construction of such a library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. Using CRISPRmass, we generated 5551 UAS-cDNA/ORF constructs covering 83% of the Drosophila genes conserved in humans in the Drosophila Genomics Resource Center (DGRC) Gold Collection, and among them, 5518 were generated within 3 mo by three people. Our results show that CRISPRmass allows modulization, simplicity, efficiency, and adaptability in the generation of a genome-wide UAS-cDNA/ORF plasmid library by using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various genome-wide libraries in general and is an alternative to Gateway technology in high-throughput plasmid library editing. Furthermore, the more than 5500 UAS-cDNA/ORF plasmids of Drosophila genes serve as a powerful resource for gain-of-function (GOF) screening in cultured cells and for generation of a transgenic UAS-cDNA/ORF library in Drosophila.

APEX, a Master Key To Resolve Membrane Topology in Live Cells.

Determining the topology of the membrane proteome is fundamental for understanding its function at the membrane. However, conventional methods involving test tube reactions often lead to unreliable results, which do not accurately reflect membrane topology under physiological conditions, as perturbations occur during lysis. In this Perspective, we introduce a new method using engineered ascorbate peroxidase (APEX) for revealing membrane topological information in live cells without performing complicated sample preparation. We also discuss several examples of clearly resolved membrane topologies of various important mitochondrial proteins (e.g., LETM1, NDUFB10, MCU, SFXN1, and EXD2) and endoplasmic reticulum proteins (e.g., HMOX1) determined by using APEX-based methods.

Synthesis of Infectious Bacteriophages in an <em>E. coli-</em>based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Phytochemical Screening and Antimicrobial Activity of Leaf Extracts of Amomum nilgiricum (Thomas) (Zingiberaceae) from Western Ghats, India

Amomum nilgiricum (Thomas) is a new species reported from the Silent Valley National Park, Western Ghats of Kerala. In the present study, qualitative and quantitative estimation of phytochemicals present in leaf extracts were carried out by test tube reactions and spectro-photometric methods. Methanolic leaf extract showed highest yield of alkaloids, saponins, phenolics, steroids, flavonoids and tannins with 34.63 ± 0.5 mgAE/g, 38.52 ± 0.017 mg DE/g, 95.4 ± 1.19 mg GAE/g, 12.51 ± 0.89 mg CE/g, 47.48 ± 0.15 mg QE/g and 29.97 ± 2.21 mg TAE/g respectively. Solvent extracts were screened for antimicrobial activities against human pathogenic bacteria and fungi by agar well diffusion method. Maximum zone of inhibition was observed in ethyl acetate extract and methanolic extract against Staphylococcus aureus (22.74 mm) and Aspergillus flavus (14.73 mm) respectively at 100 mg/mL. The minimum inhibitory concentrations (MICs) of different extracts against all pathogens ranged between 1.25 to 10 mg/mL. The MIC values of methanolic extract for antifungal efficacy against all tested fungi were found to be at 5 mg/mL.

Red Blood Cell-Encapsulation of L-Asparaginase Favorably Modulates Target Selectivity and Pharmacodynamics

L-asparaginase (L-ASP) is an enzyme-drug that has been used for decades to treat acute lymphoblastic leukemia (ALL). The red blood cell (RBC)-encapsulated L-ASP product GRASPA (eryaspase) was introduced recently with the goal of reducing L-ASP side effects without compromising efficacy. We previously showed in a Phase 2/3 trial that none of the patients treated with GRASPA had evidence of allergic reactions during induction compared to 46% of patients treated with non-encapsulated L-ASP (Baruchel et al., ASH, 2015). Similarly, evidence of pancreatic or hepatic toxicities were substantially lower with GRASPA compared to native L-ASP. Hence, GRASPA exhibits a clear benefit over L-ASP in terms of toxicity profile.The next critical question is whether GRASPA can maintain asparagine depletion. Since plasma asparagine must be transported into the RBC to be degraded by GRASPA, the RBC membrane could limit the efficacy of GRASPA. Therefore, we sought to characterize the transport and degradation of amino acids between the plasma and RBC cytoplasm in the presence of L-ASP or GRASPA. To that end, we used a bioanalytical method for the simultaneous analysis of the metabolites asparagine, aspartic acid, glutamine, and glutamic acid. Notably, the method completely quenches L-ASP and GRASPA and uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to achieve high sensitivity, accuracy, and precision. The method also involves correction for ex vivo (post-sampling) depletion of target amino acids, which occurs, for example, during centrifugation of whole blood to separate RBCs and plasma. Specifically, we added two stable isotope-labeled amino acids, 13C4-asparagine and 13C5-glutamine, to the sample at the point of collection. After centrifugation, samples were quenched by addition of 10% formic acid. Concentrations of all 12C- amino acids and their 13C- counterparts were then determined by LC-MS/MS. The difference between the nominal concentrations of the 13C amino acid substrates and their L-ASP-generated products were used to correct for the actual concentrations of the 12C amino acids. In that manner, measuring ex vivo conversion of 13C4-asparagine to 13C4-aspartate and conversion of 13C5-glutamine to 13C5-glutamate provided correction factors to calculate the original concentrations of the endogenous metabolites (unlabeled asparagine and glutamine) present in the sample at the time of collection.In whole blood test tube reactions containing L-ASP or GRASPA at matched overall asparaginase activities, the glutaminase activity of GRASPA was 10-fold lower than that of L-ASP, with initial rates of approximately 0.8 µM/min and 8.0 µM/min, respectively. Therefore, GRASPA exhibits significantly decreased glutaminase activity relative to L-ASP, resulting in an approximately 10-fold increase in selectivity for asparagine over glutamine, which may explain the observed decrease in frequency of adverse events in clinical trials with GRASPA compared to L-ASP. Notably, in the presence of GRASPA, asparagine was rapidly and extensively converted to aspartic acid inside the RBC of GRASPA, whereas no aspartic acid accumulated in the RBC following treatment of whole blood with L-ASP.In conclusion, the RBC membrane of GRASPA imbues L-ASP with improved target selectivity, which may explain the better toxicity profile of GRASPA. Altered target selectivity has been added to a growing list of beneficial properties of the RBC membrane, including improved half-life and decreased immunogenicity. [Display omitted] DisclosuresLorenzi:Erytech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. Weinstein:NIH: Patents & Royalties: patent related to L-asparaginase. Swart:Erytech Pharma: Employment, Membership on an entity’s Board of Directors or advisory committees. El-Hariry:Erytech Pharma: Employment.

Jatropha curcas L: Phytochemical, antimicrobial and larvicidal properties

ObjectiveTo evaluate antimicrobial activities as well as the phytochemical and lavicidal properties of different parts of Jatropha curcas L. (J. curcas) growing in Mauritius. MethodsDetermination of the presence of phytochemicals in the crude plants extracts by test tube reactions. Disc diffusion method and microdilution method were used to detect the antimicrobial sensitivity and activity (minimal inhibitory concentration). The crude solvent extracts were also tested on the larvae of two insects, Bactrocera zonata and Bactrocera cucurbitae (Diptera, Tephritidae). ResultsThe antimicrobial activities were significantly dependent for the different crude plant extracts on the thirteen microorganisms tested. For the Gram-positive bacteria, the crude ethyl acetate extract was more efficient compared to the Gram-negative bacteria with both solvents being effective. The crude ethyl acetate extract of J. curcas bark and mature seed oil showed the highest efficacy. The highest mortality percentage was observed after 24 h for both Diptera flies with (66.67 ± 2.89)% of Bactrocera cucurbitae larvae killed by ethyl acetate extract of J. curcas bark. ConclusionsThis paper compared the different J. curcas plant sections with respect to the effectiveness of the plant as a potential candidate for new pharmaceuticals. The larvicidal effect was also studied in order to demonstrate the dual purpose of the plant.

Cell-Free Expression Systems: From Gene Circuits to Self-Assembly Processes in a Test Tube

Cell-free transcription-translation (TX-TL) systems are becoming powerful platforms to construct complex biological systems in vitro through the expression of DNA programs. Considerable efforts have been made to improve those systems in the past decade. Our laboratory has developed the most efficient and versatile in vitro E. coli TX-TL system to express synthetic or natural DNA programs encoding for self-assembly processes. Either in test tube reactions, in emulsion droplets or in liposomes, cell-free TX-TL reactions are now used for research studies ranging from elementary gene circuits to minimal cells.I will present our custom-made cell-free TX-TL system, its current capabilities and limitations. Recently we have shown that entire phages can be synthesized in cell-free TX-TL reactions from their genomes. The bacteriophage T7, composed of about 60 genes, is entirely synthesized in a single test tube reaction from its 40 kbp genomic DNA. Replication of the T7 DNA instructions occurs concurrently with phage expression and self-assembly. The phage phiX174 composed of a dozen of genes can also be synthesized from its genome. Encapsulated inside cell-sized phospholipid liposomes, the TX-TL system is used to construct a prototype of minimal cell. I will present this cell-free synthetic biology platform, our last experiments and how this system can be used to study the relationship between information and self-organization.

Efficient and scalable method for scaling up cell free protein synthesis in batch mode

A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.

Potassium Tris(oxalato)ferrate(III): A Versatile Compound To Illustrate the Principles of Chemical Equilibria

Using a simple coordination compound, such as potassium tris(oxalato)ferrate(III), a series of test-tube reactions are proposed that allow students to visualize concepts such as equilibrium, stability, and solubility, as well as the physical constants associated with these concepts (equilibrium constant, stability constant, and solubility product). Displacement of the equilibria involved, depending on the quantity and properties of the reagents used, gives students a picture of how chemistry depends on equilibria processes.

Quantitative determination of amino acids in protein hydrolyzates by thin-layer chromatography

A micro method for the quantitative determination of amino acids in protein hydrolyzates has been proposed which permits the analysis of two hydrolyzates to be performed in 4–5 h. After the separation of the amino acids by TLC in cellulose, they are determined quantitatively by the colorimetric method using a MKMF-1 microphotocolorimeter. Proline and tryptophan are determined separately with the aid of specific test-tube reactions. The sensitivity of the method permits the analysis of 10–15 µg of aminoacid mixture.

School science — 5

School Science 4, February issue, dealt with a first lesson in crystallography. In this issue Russell Edwards indicates how he can demonstrate test tube reactions using a slide projector.

Teaching principles of quantitative analysis with "qual" reactions

A series of test-tube reactions using semi-micro techniques has been devised to precede each section of the course in quantitative analysis designed to illustrate qualitatively or semi-qualitatively the chemical reactions or principles that are the basis of each quantitative exercise.

METAL REDUCTION REACTIONS OF The MELANINS: SILVER AND FERRIC FERRICYANIDE REDUCTION BY VARIOUS REAGENTS IN VITRO

Test tube reactions of 163 reagents with silver solutions are recorded in tabular form. The silver solutions used were silver nitrate at pH 4 and at pH 6, ammoniacal silver nitrate, all at about 25°C., and Gomori's methenamine silver at 60°C. Ferric ferricyanide reduction is also included in the tables for comparison. Of the substances tested 30 quite promptly reacted with acid silver nitrate as well as the other reagents, 28 gave delayed reactions with acid, prompt with alkaline silver, 24 gave variable acid, and delayed alkaline silver reactions, 27 gave no reaction with acid silver and delayed reactions with alkaline, 16 reacted slowly with hot methenamine silver and not or scarcely at all with cold ammoniacal silver. The remaining 38 reactants did not blacken with silver solutions. Also tabulated are the pigment formation reactions of 13 polyphenols, indoles and sulfur amino acids with chromic acid, potassium bichromate, potassium chromate, periodic acid, sodium iodate and potassium chlorate.

Relation of Anaphylaxis to Immunity

Summary and Conclusions The blood of an actively immunized dog, therefore, contains no antibody that serves as a demonstrable circulating defense to hypersensitize fixed tissues. The immune blood, however, does contain a desensitizing substance, capable of causing complete desensitization of hypersensitive fixed tissues. This desensitization is effected only after a latent period of about forty-eight hours. All phenomena of passive desensitization or passive immunization thus far studied in dogs may be accounted for as a result of the action of this desensitizing antibody. As pointed out in previous papers (2, 4), there is conclusive evidence that in dogs the sensitizing antibody and desensitizing antibody are of different chemical composition, and that neither of these substances is identical with the specific precipitin of our test tube reactions.