Chromosomal Damage, Chromosome Instability, and Polymorphisms in GSTP1 and XRCC1 as Biomarkers of Effect and Susceptibility in Farmers Exposed to Pesticides.

In the department of Boyacá, Colombia, agriculture stands as one of the primary economic activities. However, the escalating utilization of pesticides within this sector has sparked concern regarding its potential correlation with elevated risks of genotoxicity, chromosomal alterations, and carcinogenesis. Furthermore, pesticides have been associated with a broad spectrum of genetic polymorphisms that impact pivotal genes involved in pesticide metabolism and DNA repair, among other processes. Nonetheless, our understanding of the genotoxic effects of pesticides on the chromosomes (as biomarkers of effect) in exposed farmers and the impact of genetic polymorphisms (as susceptibility biomarkers) on the increased risk of chromosomal damage is still limited. The aim of our study was to evaluate chromosomal alterations, chromosomal instability, and clonal heterogeneity, as well as the presence of polymorphic variants in the GSTP1 and XRCC1 genes, in peripheral blood samples of farmers occupationally exposed to pesticides in Aquitania, Colombia, and in an unexposed control group. Our results showed statistically significant differences in the frequency of numerical chromosomal alterations, chromosomal instability, and clonal heterogeneity levels between the exposed and unexposed groups. In addition, we also found a higher frequency of chromosomal instability and clonal heterogeneity in exposed individuals carrying the heterozygous GSTP1 AG and XRCC1 (exon 10) GA genotypes. The evaluation of chromosomal alterations and chromosomal instability resulting from pesticide exposure, combined with the identification of polymorphic variants in the GSTP1 and XRCC1 genes, and further research involving a larger group of individuals exposed to pesticides could enable the identification of effect and susceptibility biomarkers. Such markers could prove valuable for monitoring individuals occupationally exposed to pesticides.

DNA methylases for site-selective inhibition of type IIS restriction enzyme activity.

DNA methylases of the restriction-modifications (R-M) systems are promising enzymes for the development of novel molecular and synthetic biology tools. Their use in vitro enables the deployment of independent and controlled catalytic reactions. This work aimed to produce recombinant DNA methylases belonging to the R-M systems, capable of in vitro inhibition of the type IIS restriction enzymes BsaI, BpiI, or LguI. Non-switchable methylases are those whose recognition sequences fully overlap the recognition sequences of their associated endonuclease. In switch methylases, the methylase and endonuclease recognition sequences only partially overlap, allowing sequence engineering to alter methylation without altering restriction. In this work, ten methylases from type I and II R-M systems were selected for cloning and expression in E. coli strains tolerant to methylation. Isopropyl ß-D-1-thiogalactopyranoside (IPTG) concentrations and post-induction temperatures were tested to optimize the soluble methylases expression, which was achieved with 0.5 mM IPTG at 20 °C. The C-terminal His6-Tag versions showed better expression than the N-terminal tagged versions. DNA methylation was analyzed using purified methylases and custom test plasmids which, after the methylation reactions, were digested using the corresponding associated type IIS endonuclease. The non-switchable methylases M2.Eco31I, M2.BsaI, M2.HpyAII, and M1.MboII along with the switch methylases M.Osp807II and M2.NmeMC58II showed the best activity for site-selective inhibition of type IIS restriction enzyme activity. This work demonstrates that our recombinant methylases were able to block the activity of type IIS endonucleases in vitro, allowing them to be developed as valuable tools in synthetic biology and DNA assembly techniques. KEY POINTS: • Non-switchable methylases always inhibit the relevant type IIS endonuclease activity • Switch methylases inhibit the relevant type IIS endonuclease activity depending on the sequence engineering of their recognition site • Recombinant non-switchable and switch methylases were active in vitro and can be deployed as tools in synthetic biology and DNA assembly.

Análisis molecular y filogenético del genotipo F de Chlamydia trachomatis aislados en México en 2011 / Molecular and phylogenetic analysis of the F genotype of Chlamydia trachomatis isolated in Mexico in 2011

Resumen Antecedentes: Chlamydia trachomatis es la bacteria que se detecta con mayor frecuencia en las infecciones de transmisión sexual. Se han identificado 20 genotipos de C. trachomatis mediante el gen ompA y varias genovariantes mediante el análisis de polimorfismo de un solo nucleótido (SNP). En México, el genotipo F es el más frecuente. Objetivo: Identificar la existencia de subtipos del genotipo F. Método: Se analizaron siete cepas del genotipo F de C. trachomatis aisladas en 2011, mediante secuenciación de nucleótidos y mapeo con enzimas de restricción. Resultados: El análisis de SNP mostró dos cepas con el mismo SNP en el nucleótido 288 (C288T), mientras que con enzimas de restricción se identificó una variante con diferente RFLP (polimorfismo de la longitud de fragmentos de restricción) cuando se tratan con la mezcla de enzimas HinfI y TaqI. Conclusión: En México se encuentran dos subtipos del genotipo F y solo las enzimas de restricción HinfI y TaqI pueden identificar la existencia de uno de estos genotipos F.

Abstract Background: Chlamydia trachomatis is the most frequently identified bacterium in sexually transmitted infections. Twenty C. trachomatis genotypes have been determined using the ompA gene and several genovariants by single nucleotide polymorphism (SNP) analysis. In Mexico, the F genotype is the most frequent. Objective: To identify subtypes of the F genotype. Method: Seven C. trachomatis genotype F strains isolated in 2011 were analyzed by nucleotide sequencing and restriction enzyme mapping. Results: SNP analysis showed two strains with the same SNP at nucleotide 288 (C288T), while with res-triction enzymes, a variant with different RFLP (restriction fragment length polymorphism) was identified when treated with the mixture of HinfI and TaqI enzymes. Conclusion: In Mexico, there are two subtypes of F, and only with restriction enzymes HinfI and TaqI can identify one of the genovariants of the F genotype.

Polymorphism and mutational diversity of virulence (vcgCPI/vcgCPE) and resistance determinants (aac(3)-IIa, (aacC2, strA, Sul 1, and 11) among human pathogenic Vibrio species recovered from surface waters in South-Western districts of Uganda.

BACKGROUND: Vibrio species are among the autochthonous bacterial  populations found in surface waters and associated with various life-threatening extraintestinal diseases, especially in human populations with underlying illnesses and wound infections. Presently, very diminutive information exists regarding these species' mutational diversity of virulence and resistance genes. This study evaluated variations in endonucleases and mutational diversity of the virulence and resistance genes of Vibrio isolates, harboring virulence-correlated gene (vcgCPI), dihydropteroate synthase type 1 and type II genes (Sul 1 and 11), (aadA) aminoglycoside (3'') (9) adenylyltransferase gene, (aac(3)-IIa, (aacC2)a, aminoglycoside N(3)-acetyltransferase III, and (strA) aminoglycoside 3'-phosphotransferase resistance genes. METHODS: Using combinations of molecular biology techniques, bioinformatics tools, and sequence analysis. RESULTS: Our result revealed various nucleotide variations in virulence determinants of V. vulnificus (vcgCPI) at nucleotide positions (codon) 73-75 (A → G) and 300-302 (N → S). The aminoglycosides resistance gene (aadA) of Vibrio species depicts a nucleotide difference at position 482 (A → G), while the aminoglycosides resistance gene (sul 1 and 11) showed two variable regions of nucleotide polymorphism (102 and 140). The amino acid differences exist with the nucleotide polymorphism at position 140 (A → E). The banding patterns produced by the restriction enzymes HinP1I, MwoI, and StyD4I showed significant variations. Also, the restriction enzyme digestion of protein dihydropteroate synthase type 1 and type II genes (Sul 1 and 11) differed significantly, while enzymes DpnI and Hinf1 indicate no significant differences. The restriction enzyme NlaIV showed no band compared to reference isolates from the GenBank. However, the resistant determinants show significant point nucleotide mutation, which does not produce any amino acid change with diverse polymorphic regions, as revealed in the restriction digest profile. CONCLUSION: The described virulence and resistance determinants possess specific polymorphic locus relevant to pathogenomics studies, pharmacogenomic, and control of such water-associated strains.

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures.

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site-specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to "smart" functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

Evaluating restriction enzyme selection for reduced representation sequencing in conservation genomics.

Conservation genomic studies in non-model organisms generally rely on reduced representation sequencing techniques based on restriction enzymes to identify population structure as well as candidate loci for local adaptation. While the expectation is that the reduced representation of the genome is randomly distributed, the proportion of the genome sampled might depend on the GC content of the recognition site of the restriction enzyme used. Here, we evaluated the distribution and functional composition of loci obtained after a reduced representation approach using Genotyping-by-Sequencing (GBS). To do so, we compared experimental data from two endemic fish species (Symphodus ocellatus and Symphodus tinca, EcoT22I enzyme) and two ecosystem engineer sea urchins (Paracentrotus lividus and Arbacia lixula, ApeKI enzyme). In brief, we mapped the sequenced loci to the phylogenetically closest reference genome available (Labrus bergylta in the fish and Strongylocentrotus purpuratus in the sea urchin datasets), classified them as exonic, intronic and intergenic, and studied their function by using Gene Ontology (GO) terms. We also simulated the effect of using both enzymes in the two reference genomes. In both simulated and experimental data, we detected an enrichment towards exonic or intergenic regions depending on the restriction enzyme used and failed to detect differences between total loci and candidate loci for adaptation in the empirical dataset. Most of the functions assigned to the mapped loci were shared between the four species and involved a myriad of general functions. Our results highlight the importance of restriction enzyme selection and the need for high-quality annotated genomes in conservation genomic studies.

AURKA Gene Variants rs1047972, and rs8173 Are Associated With Breast Cancer.

PURPOSE: Association between variants rs1047972 and rs8173 of the AURKA gene in healthy women and breast cancer (BC) in a Mexican population. METHODS: Genomic DNA samples from 409 healthy women and 572 patients with BC were analyzed for variants rs1047972 and rs8173 of the AURKA gene by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: TT genotype (odds ratio [OR], 2.5; 95% confidence interval [CI], 1.22-5.11; p = 0.0015) and the T allele (OR, 1.16; 95% CI, 1.23-2.12; p = 0.0007) of the rs1047972 variant were associated as risk susceptibility for BC relative to the control group. Contrarily, the GG genotype (OR, 0.64; 95% CI, 0.43-0.94; p = 0.029) was associated as a protective factor of susceptibility of BC of the variant rs8173 of the AURKA gene. Differences were observed in the patients with BC who were carriers of the CT genotype of the rs1047972 variant with overweight, obesity, estrogen receptor-positive plus obesity, Ki-67 (≥ 20%) plus history familial positive of cancer; and for variant rs8173 the BC patients who were CG carriers and presented chemotherapy gastric toxicity, hormonal receptor positive plus chemotherapy gastric toxicity, and menopause status plus chemotherapy gastric toxicity (p < 0.05). Two common haplotypes were identified in the study groups: CG and TC genotypes, were associated as a protective and risk factor, respectively (p < 0.05). CONCLUSION: Variants rs1047972 and rs8173 of the AURKA gene and the TC haplotype were associated as risk susceptibility factors for BC in this population.

Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning.

Megaprimer-based polymerase chain reaction (PCR) strategies allow the versatile and fast assembly and amplification of a myriad of tailor-made or random DNA sequences readily available for conventional or restriction-free (RF) cloning.In this chapter, we present a megaprimer-based PCR protocol that enables the expeditious construction of customized fusion genes ready for cloning into commercial expression plasmids. With the expanding use of protein tag technology in the most diverse application fields, this protocol remains a versatile and affordable solution for the synthesis and fusion of peptide tags/domains of interest.

pHAPE: a plasmid for production of DNA size marker ladders for gel electrophoresis.

DNA size markers (also known as 'molecular weight markers' or 'DNA ladders') are an essential tool when using gel electrophoresis to identify and purify nucleic acids. However, the cost of these DNA ladders is not insignificant and, over time, impinges on the funds available for research and training in molecular biology. Here, we describe a method for the generation of 'pHAPE', a plasmid from which a variety of DNA ladders can be generated via simple restriction enzyme digestions. The pHAPE plasmid can be generated by mutagenesis of the commonly used pBluescript II SK+ phagemid followed by insertion of a 7141 bp sequence (comprised of three smaller, synthetic fragments). Our use of pHAPE allows us some small relief from the ever-rising costs of performing molecular biology experiments ('Don't worry, pHAPE').

A CRISPR-based approach using dead Cas9-sgRNA to detect SARS-CoV-2.

Rapid, highly specific, and robust diagnostic kits to detect viruses and pathogens are needed to control disease spread and transmission globally. Of the many different methods proposed to diagnose COVID-19 infection, CRISPR-based detection of nucleic acids tests are among the most prominent. Here, we describe a new way of using CRISPR/Cas systems as a rapid and highly specific tool to detect the SARS-CoV-2 virus using the in vitro dCas9-sgRNA-based technique. As a proof of concept, we used a synthetic DNA of the M gene, one of the SARS-CoV-2 virus genes, and demonstrated that we can specifically inactivate unique restriction enzyme sites on this gene using CRISPR/Cas multiplexing of dCas9-sgRNA-BbsI and dCas9-sgRNA-XbaI. These complexes recognize and bind to the target sequence spanning the BbsI and XbaI restriction enzyme sites, respectively, and protect the M gene from digestion by BbsI and/or XbaI. We further demonstrated that this approach can be used to detect the M gene when expressed in human cells and from individuals infected with SARS-CoV-2. We refer to this approach as dead Cas9 Protects Restriction Enzyme Sites, and believe that it has the potential to be applied as a diagnostic tool for many DNA/RNA pathogens.

The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Identification of Leishmania species and frequency distribution of LRV1 and LRV2 viruses on cutaneous leishmaniasis patients in Isfahan Province, Iran.

PURPOSE: Leishmaniasis is one of the most serious health problems in developing countries. Iran is one of the endemic regions of cutaneous leishmaniasis. Leishmania RNA virus (LRV) is a dsRNA virus member of the Totiviridae family, which was first detected in the promastigotes of Leishmania braziliensis guyanensis. Our study aimed to investigate possible changes in the predominant and causative strains of CL and screening the LRV1 and LRV2 species genome from Leishmania species isolated from the lesions of patients. MATERIALS AND METHODS: Direct smear samples obtained from 62 patients with leishmaniasis referring to the Skin Diseases and Leishmaniasis Research Center in Isfahan province during 2021-2022 were examined. Total DNA extraction procedures and conservation of site-specific multiplex PCR and nested PCR were performed for detecting Leishmania species. The molecular identification of LRV1 and LRV2 viruses, samples were used for total RNA extraction and real-time (RT)-PCR analysis, followed by conducting a restriction enzyme assay to confirm the PCR products. RESULTS: Of the total Leishmania isolates, 54 and 8 isolates were identified as L. major and L. tropica, respectively. LRV2 was identified in 18 samples affected by L. major, while LRV1 was only detected in one of the samples with L. tropica. No LRV2 was found in any samples with L. tropica. The results showed that there was a significant relationship between LRV1 and the type of leishmaniasis (Sig. â€‹= â€‹0.009, P â€‹≤ â€‹0.05), while this relationship was not observed between LRV2 and the type of leishmaniasis. CONCLUSIONS: The presence of a significant number of LRV2 in isolated samples, as well as the recognition of LRV1 in one of the Old World leishmaniasis species, which is a new result, could pave the way for investigating further aspects of this disease and successful treatment strategies in future studies.

Computational Protocol for DNA Methylation Profiling in Plants Using Restriction Enzyme-Based Genome Reduction.

Epigenetics can be described as heritable phenotype changes that do not involve alterations in the underlying DNA sequence. Having widespread implications in fundamental biological phenomena, there is an increased interest in characterizing epigenetic modifications and studying their functional implications. DNA methylation, particularly 5-methylcytosine (5mC), stands out as the most studied epigenetic mark and several methodologies have been created to investigate it. With the development of next-generation sequencing technologies, several approaches to DNA methylation profiling were conceived, with differences in resolution and genomic scope. Besides the gold standard whole-genome bisulfite sequencing, which is costly for population-scale studies, genomic reduced representation methods emerged as viable alternatives to investigate methylation loci. Whole-genome bisulfite sequencing provides single-base methylation resolution but is costly for population-scale studies. Genomic reduction methods emerged as viable alternatives to investigate a fraction of methylated loci. One of such approaches uses double digestion with the restriction enzymes PstI and one of the isoschizomers, MspI and HpaII, with differential sensitivity to 5mC at the restriction site. Statistical comparison of sequencing reads counts obtained from the two libraries for each sample (PstI-MspI and PstI-HpaII) is used to infer the methylation status of thousands of cytosines. Here, we describe a general overview of the technique and a computational protocol to process the generated data to provide a medium-scale inventory of methylated sites in plant genomes. The software is available at https://github.com/wendelljpereira/DArTseqMet .

Characterization of BoLA class II DQA and DQB by PCR-RFLP, cloning, and sequencing reveals sequence diversity in crossbred cattle.

The BoLA class II DQA and DQB genes in crossbred cattle were studied using PCR-RFLP, cloning, and sequencing techniques. Seventy-two crossbred cattle (Vrindavani) were used in the current study. HaeIII and XbaI restriction enzymes digested DQA exon 2-3, revealing seven (HaeIII-A-G) and three (XbaI A-C) motifs, respectively. The BoLA-DQB gene was analyzed using PCR-RFLP with PstI and TaqI restriction enzymes, yielding five restriction motifs for each restriction enzyme (PstI-A-E and TaqI-A-E). In crossbred cattle, addition, deletion, and substitutions were observed in distinct sequences, resulting in variations in overall gene length. Changes in nucleotides at positions 64-80, 110-200, and 207-264 were largely responsible for polymorphism in DQA exon 2. The phylogenetic analysis predicted a high degree of nucleotide and amino acid changes in DQA exon 2-3 and DQB exon 2. DQA genes had a nucleotide dissimilarity of 0.3-25.4 percent, while DQB genes had a nucleotide dissimilarity of 1.5-14.3 percent. We cloned and sequenced 20 genotypes based on PCR-RFLP of the DQA and DQB genes. The current study observed variation in the DQA and DQB genes and will serve as a foundation for future research on the BoLA DQA and DQB genes.

Application of 16S rRNA virtual RFLP for the discrimination of some closely taxonomic-related lactobacilli species.

BACKGROUND: Several species in Lactobacillaceae family were recognized as potential probiotic bacteria. In this group of lactic acid bacteria, species are taxonomically closed and usually share similar 16S rRNA gene, thus, instead of so their identification and discrimination are too difficult. METHOD: In the present study, virtual restriction fragment length polymorphism (RFLP) is instead of was used as a tool to discriminate between the closely related species Lactiplantibacillus plantarum (L plantarum), Lactiplantibacillus paraplantarum (L paraplantarum), and Lactiplantibacillus pentosus (L pentosus); Latilactobacillus sakei (L sakei), Latilactobacillus curvatus(L curvatus), and Latilactobacillus graminis (L graminis); Lacticaseibacillus casei (L casei), Lacticaseibacillus paracasei (L paracasei), Lacticaseibacillus zeae, and Lacticaseibacillus rhamnosus; Lactobacillus gasseri (L gasseri) and Lactobacillus johnsonii (L johnsonii). In silico comparative analysis of 16S rRNA sequences digested by 280 restriction enzymes was performed in order to search the key enzymes which gives different profiles. RESULTS: Results revealed that L casei, L paracasei, L zeae, and Lb rhamnosus could be separated from each other on the basis of AlwI, BpuEI, BsgI, BsrDI, BstYI, EarI, MluCI, and NsPI RFLP. Results showed also that different RFLP patterns were obtained from L sakei, L graminis and L curvatus by using both AflI and NspI endonucleases (in separated restriction) and L plantarum, L paraplantarum, and L pentosus were distinguished each one from the other by MucI, NspI, and TspDTI PCR-RFLP. Lb gasseri and L johnsonii were also separated on the basis of Mse I, Taq I, and Dra I RFLP. CONCLUSION: In this study, we proved that too closely related species could be separated in virtual analysis on basis of their 16S rRNA RFLP patterns using key restriction enzymes method.

Genotyping by Sequencing Advancements in Barley.

Barley is considered an ideal crop to study cereal genetics due to its close relationship with wheat and diploid ancestral genome. It plays a crucial role in reducing risks to global food security posed by climate change. Genetic variations in the traits of interest in crops are vital for their improvement. DNA markers have been widely used to estimate these variations in populations. With the advancements in next-generation sequencing, breeders could access different types of genetic variations within different lines, with single-nucleotide polymorphisms (SNPs) being the most common type. However, genotyping barley with whole genome sequencing (WGS) is challenged by the higher cost and computational demand caused by the large genome size (5.5GB) and a high proportion of repetitive sequences (80%). Genotyping-by-sequencing (GBS) protocols based on restriction enzymes and target enrichment allow a cost-effective SNP discovery by reducing the genome complexity. In general, GBS has opened up new horizons for plant breeding and genetics. Though considered a reliable alternative to WGS, GBS also presents various computational difficulties, but GBS-specific pipelines are designed to overcome these challenges. Moreover, a robust design for GBS can facilitate the imputation to the WGS level of crops with high linkage disequilibrium. The complete exploitation of GBS advancements will pave the way to a better understanding of crop genetics and offer opportunities for the successful improvement of barley and its close relatives.

Synthetic Assembly DNA Cloning of Multiplex Hextuple Luciferase Reporter Plasmids.

Multiplex hextuple luciferase assaying allows monitoring the activity of five experimental pathways against one control at the same time. To perform multiplex hextuple luciferase assaying, six orthogonal luciferase reporter units are needed of which five are pathway-specific and one acts as a control for normalization. To ensure stoichiometric delivery of all six luciferase reporters in every transfected cell, synthetic assembly DNA cloning is used to stitch together all six luciferase reporter units into a single vector. Here, we provide a detailed three-step synthetic assembly DNA protocol to generate multiplex hextuple luciferase reporter plasmids for any five cellular signaling pathways of interest, against a control normalization pathway. A first protocol is provided on how to generate plasmids that contain novel transcription factor-binding motifs for specific transcription factors. A second protocol details on how to couple these novel transcription factor-binding motifs to one of five orthogonal luciferases to obtain specific luciferase reporters for cellular signaling pathways acting upstream of those transcription factor-binding motifs. Finally, a third protocol provides details on how to assemble orthogonal luciferase reporters for five cellular signaling pathways acting upstream of five unique transcription factor-binding motifs together with a control constitutive pathway luciferase reporter that will be used for normalization to obtain a final multiplex hextuple luciferase vector.

Morphogenesis and Genetic Diversity of Some Virulent Phages Specific for Bacillus velezensis.

<b>Background and Objective:</b> Phosphorus (P) is one of the most limiting nutrients for plant growth. Phosphorus deficiency is limiting crop production in many agricultural soils worldwide. The application of phosphorus solubilizing bacteria (PSB) to soils can replace or partially reduce using of inorganic P fertilizers. A bacteriophage, or phage, is a virus that infects a bacterial cell, taking over the host cell's genetic material. The four phages were propagated, purified, studied for the morphological properties, finally studying the genetic diversity. <b>Materials and Methods:</b> Obtained, examined the efficiency and identification of bacteria for solubilizing phosphorus. Isolation, studying the properties and studying genetic diversity. <b>Results:</b> Four virulent phages (Bv₁, Bv₂, Bv₃ and Bv₄) specific for <i>Bacillus velezensis</i> were isolated from the Egyptian soil. The <i>Bacillus</i> phages were purified by alternative low and high-speed centrifugation methods. Electron micrographs showed that phages appeared to be a member of the <i>Siphoviridae </i>family based on their structure and particle morphology (the particles have a head and long non-contractile tail). Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique was performed to determine the properties of viral proteins. It was found that the Bv₁ virus had five structural proteins, while Bv₂ and Bv₃ virus had eight structural proteins and finally, the Bv₄ virus had ten structural proteins. The purity and quantity of isolated DNAs were determined spectrophotometrically. Data showed that the concentration of Bv₁ DNA was 0.75 µg, Bv₂ DNA and Bv₃ DNA was 0.60 µg and finally Bv₄ DNA 0.55 µg µL¹. The analysis of genetic material of <i>B. velezensis</i> phages was determined based on both the ISSR-PCR technique and the effect of restriction enzymes. Data showed different amplification patterns with all phages. <b>Conclusion:</b> The bacteriophages of <i>B. velezensis</i> were isolated from soil, propagated, purified, study some of its properties.

Case study: Perspectives on the use of LEGO® bricks in the biochemistry classroom.

The use of LEGO® bricks in the higher education classroom has increased in the last two decades. This is no different in the STEM classroom and several disciplines, including physics, chemistry, and biology, have all made use of LEGO® bricks in some way to create models for active learning activities. Currently, the discipline to make the greatest use of LEGO® bricks is chemistry; only limited examples exist in biochemistry and the molecular life sciences. Here, we present the use of a LEGO® brick modelling activity in the introductory biochemistry classroom during the teaching of metabolism. We present student comments on the activity and the models that were generated by the students. Additionally, we focus on other instructor and project student-designed models for the teaching of ATP synthesis, gene regulation and restriction digestion. Interestingly, both the gene regulation and restriction digest activities were generated with the help of undergraduate students or recent graduates, by applying a backward design approach. This case study seeks to encourage more molecular life science educators to adopt the use of LEGO® bricks in their classrooms to engage in more active learning.

Coexpression and Reconstitution of Enzymatic Cascades in Bacteria Using UbiGate.

Coexpression of multiple genes of interest (GOIs) is advantageous for many purposes including the elucidation of protein complexes, reconstitution of enzymatic cascades that mediate the biosynthesis of compounds, the study of signaling cascades, or the elucidation of posttranslational modification. Additional advantages of coexpressing proteins is increased solubility and stability of proteins. For this purpose we developed UbiGate, a modular system based on Golden Gate cloning that enables the generation of polycistronic expression cassettes. Their generation is achieved in four simple steps: (1) GOIs are amplified via PCR, (2) and restriction-ligated into level 0 cloning vectors. Next, (3) the GOIs in a level 0 vector are restriction-ligated into a dedicated set of level 1 vectors that define the position of the GOI within the operon. In the last step (4), level 1 vectors are cloned into a modified pET28-GG expression vector. The resulting modules at each step can be reused to generate fusions with different tags in any desired order and orientation, to include up to six different proteins representing a useful tool facilitating the study of plant metabolic and signaling pathways.