scholarly journals Probe design for exploiting gene encoding pectinesterase from dna metagenome data of bacteria in goat rumen and co-expression of gpecs1 gen with chaperone pg-kje8 in Escherichia coli

2018 ◽  
Vol 40 (1) ◽  
pp. 84-91
Author(s):  
Nguyen Khanh Hoang Viet ◽  
Do Thi Huyen ◽  
Le Tung Lam ◽  
Phung Thi Lan ◽  
Phung Thu Nguyet ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Dna Sequences ◽  
Soluble Form ◽  
Probe Design ◽  
Sequencing Data ◽  
Gene Encoding ◽  
E Coli ◽  
Environmental Treatment ◽  
Query Coverage ◽  
Goat Rumen

This article introduces the steps of constructing and using probe to exploit the gene encoding pectinesterase from metagenome DNA sequencing data by next generation gene sequencing tools. Probe was used to exploit and select the gene encoding for pectinesterase from the metagenome DNA sequences of bacteria in goat rumen and thereby select a sequence to express in E. coli. According to the CAZy classification system, pectinesterase belongs to the family of carbohydrates esterases CE8 is an enzyme that has many applications in the food processing industry, environmental treatment, animal feed processing and medicine. As the results, 3 sequences of CE8 was retrieved from CAZy database and one probe was designed, this probe length was 367 amino acids contained all the conserved amino acid residues: 200 conserved residues in all sequence, 72 residues similar in almost sequences and residues conserved in many sequences and homologus; choosed highest alkalinity index. Using the probe designed, we filtered four coding sequences for pectinesterase from metagenome DNA sequencing data of bacteria in goat rumen. Spatial structure estimation with Phyre2 has only one sequencing (code 46301) with 100% sequence identity and 90% query coverage with pectinesterase. A artificial gene were synthesized and inserted into the vector pET22b (+) at the NcoI, XhoI to co-express with chaperone pG-KJE8 in E. coli. The recombinant pectinesterase enzyme is expressed in soluble form and has a pectin substrate biodegradation activity. The results demonstrate that using probe for gene extraction is feasible.

scholarly journals 1127. Genomic Analysis of Biofilm-Forming Enteroinvasive E. coli Emergent Pathogen

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S337-S338
Author(s):  
Oscar Gomez-Duarte ◽  
Julio Guerra ◽  
Ricky Ko
Keyword(s):  
Virulence Factors ◽  
Dna Sequences ◽  
Horizontal Transfer ◽  
Antibiotic Resistance Genes ◽  
Genomic Analysis ◽  
Coli Strain ◽  
Virulence Plasmid ◽  
E Coli ◽  
Query Coverage

Abstract Background Enteroinvasive Escherichia coli (EIEC) are involved in dysenteric diarrhea among children in low- and middle-income countries. EIEC strains isolated in Colombia, South America were shown to form biofilms and to be invasive in vitro. The O96:H19 serotypes and biofilm formation (BF) are not common phenotypes among EIEC, and the role they may play in diarrhea is at present unknown. The main goal of this study was to identify virulence and BF genes from EIEC genomic data. We hypothesize that EIEC O96:H19 strain 52.1 originated from horizontal transfer of a Shigella-like virulence plasmid into a non-EIEC pathogenic E coli strain. Methods WGS was performed on the BF-EIEC 52.1 strain using NextGen Illumina and Pacific Biosciences (PacBio) platforms. Publically available genomes from other EIEC O96H19 and Shigella genomes previously published were analyzed using online available software and databases including NCBI, BLAST, Mauve, among others. This analysis was tailored to identify virulence factors from the virulence factor database (VFDB). BLASTn was used to determine identity and query coverage of genes encoding the Shigella virulence factors. EIEC and Shigella genomes were analyzed on a multiple genome alignment software (Mauve) to verify results from BLASTn and to determine pseudogenes. Results The genome of EIEC O96:H19 strain 52.1 was 5,193,449 bp in size, containing 5,050 coding DNA sequences (CDSs). O96:H19 strain 52.1 carries three plasmids, the invasion plasmid (pINV) contains all type 3 secretion system (TTSS) and TTSS effectors genes previously described for Shigella and EIEC O96:H19 CFSAN029787 Italian strain. Non-TTSS virulence genes were also identified, including: long polar fimbrial gene (IpfA), enterotoxin (senB), and antibiotic resistance genes. Conclusion The EIEC O96:H19 strain 52.1 genome carries TTSS genes within a virulence plasmid, protein effector genes, and enterotoxin genes known to be associated with EIEC virulence. The EIEC O96:H19 stain 52.1 is an emergent diarrheagenic pathogen likely derived from an E. coli O96:H19 strain that acquired a Shigella-like virulence plasmid by horizontal transfer. Disclosures All authors: No reported disclosures.

scholarly journals Expression of gene encoding flavonol synthase isolating from trung du xanh tea (Camellia sinensis var. macrophylla) in E. coli

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Hoang Thi Thu Yen ◽  
Vu Thi Lan ◽  
Huynh Thi Thu Hue
Keyword(s):  
Camellia Sinensis ◽  
Expression Vector ◽  
Soluble Fraction ◽  
Soluble Form ◽  
Gene Encoding ◽  
Flavonol Synthase ◽  
E Coli ◽  
Insoluble Form

Common flavonols in plants including quercetin, kaempferol and myricetin are synthesized from dihydroflavonols (dihydroquercetin-DHQ, dihydrokaempferol-DHK and dihydromyricetin-DHM) by flavonol synthase (FLS). In tea, FLS has been shown to metabolize dihydroquercetin to quercetin. The FLS gene was cloned and sequenced from the cultivated tea (Camellia sinensis var. macrophylla) in Thai Nguyen province. In this study, we presented the results of optimizing and designing an expression vector for recombinant FLS (recombinant FLS-rFLS). The FLS gene was ligated completely to the pET32a (+) vector, then expressed in E. coli Rosetta1 and Rosetta2 strain. Using 1mM IPTG to induce the expression of rFLS at 37oC, rFLS was obtained with 52.83 kDa in size and existed predominantly as insoluble form. E. coli Rosetta1 pET32a (+)_FLSproduces rFLS in the soluble fraction than E. coli Rosetta2 pET32a (+)_FLS. Next, E. coli Rosetta1 pET32a (+)_FLSwas optimized for expression at temperatures of 30oC, 23oC and 16oC (24 and 48 hours). After being induced for expression with 1mM IPTG in 48 hours and cultured at 16oC, E. coli Rosetta1 strain containing pET32a (+) FLS produced the largest amount of rFLS in the soluble form. 

scholarly journals Bacterial methionine biosynthesis

Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1571-1584 ◽  
Author(s):  
Matteo P. Ferla ◽  
Wayne M. Patrick
Keyword(s):  
Bacterial Species ◽  
Sequencing Data ◽  
Methionine Biosynthesis ◽  
Gene Encoding ◽  
Metabolic Intermediates ◽  
E Coli ◽  
True Diversity ◽  
Preceding Step ◽  
Adenosyl Methionine ◽  
Key Steps

Methionine is essential in all organisms, as it is both a proteinogenic amino acid and a component of the cofactor, S-adenosyl methionine. The metabolic pathway for its biosynthesis has been extensively characterized in Escherichia coli; however, it is becoming apparent that most bacterial species do not use the E. coli pathway. Instead, studies on other organisms and genome sequencing data are uncovering significant diversity in the enzymes and metabolic intermediates that are used for methionine biosynthesis. This review summarizes the different biochemical strategies that are employed in the three key steps for methionine biosynthesis from homoserine (i.e. acylation, sulfurylation and methylation). A survey is presented of the presence and absence of the various biosynthetic enzymes in 1593 representative bacterial species, shedding light on the non-canonical nature of the E. coli pathway. This review also highlights ways in which knowledge of methionine biosynthesis can be utilized for biotechnological applications. Finally, gaps in the current understanding of bacterial methionine biosynthesis are noted. For example, the paper discusses the presence of one gene (metC) in a large number of species that appear to lack the gene encoding the enzyme for the preceding step in the pathway (metB), as it is understood in E. coli. Therefore, this review aims to move the focus away from E. coli, to better reflect the true diversity of bacterial pathways for methionine biosynthesis.

scholarly journals Pattern Matching for DNA Sequencing Data Using Multiple Bloom Filters

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Maleeha Najam ◽  
Raihan Ur Rasool ◽  
Hafiz Farooq Ahmad ◽  
Usman Ashraf ◽  
Asad Waqar Malik
Keyword(s):  
Dna Sequencing ◽  
Dna Sequence ◽  
Pattern Matching ◽  
Dna Sequences ◽  
Sequence Data ◽  
Bloom Filters ◽  
Sequencing Data ◽  
Dna Sequence Data ◽  
Efficient Data ◽  
Improved Accuracy

Storing and processing of large DNA sequences has always been a major problem due to increasing volume of DNA sequence data. However, a number of solutions have been proposed but they require significant computation and memory. Therefore, an efficient storage and pattern matching solution is required for DNA sequencing data. Bloom filters (BFs) represent an efficient data structure, which is mostly used in the domain of bioinformatics for classification of DNA sequences. In this paper, we explore more dimensions where BFs can be used other than classification. A proposed solution is based on Multiple Bloom Filters (MBFs) that finds all the locations and number of repetitions of the specified pattern inside a DNA sequence. Both of these factors are extremely important in determining the type and intensity of any disease. This paper serves as a first effort towards optimizing the search for location and frequency of substrings in DNA sequences using MBFs. We expect that further optimizations in the proposed solution can bring remarkable results as this paper presents a proof of concept implementation for a given set of data using proposed MBFs technique. Performance evaluation shows improved accuracy and time efficiency of the proposed approach.

scholarly journals Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.

1988 ◽  
Vol 8 (12) ◽  
pp. 5417-5424 ◽  
Author(s):  
G R Banks ◽  
S Y Taylor
Keyword(s):  
Dna Sequences ◽  
Gene Copy Number ◽  
Mutant Gene ◽  
Ustilago Maydis ◽  
Gene Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Multiple Transcripts ◽  
Different Types

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

scholarly journals Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats’ Rumen by Metagenomic Deep Sequencing

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3257
Author(s):  
Trong-Khoa Dao ◽  
Thi-Huyen Do ◽  
Ngoc-Giang Le ◽  
Hong-Duong Nguyen ◽  
Thi-Quy Nguyen ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Gene Encoding ◽  
Amylolytic Enzymes ◽  
E Coli ◽  
Lignocellulose Conversion ◽  
Genes Encoding ◽  
Abundant Genus ◽  
First Time ◽  
Goat Rumen

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats’ rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5–6.5, temperature 40–50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

scholarly journals Identification of a New Class of Cytochrome P450 from a Rhodococcus sp

2002 ◽  
Vol 184 (14) ◽  
pp. 3898-3908 ◽  
Author(s):  
Gareth A. Roberts ◽  
Gideon Grogan ◽  
Andy Greter ◽  
Sabine L. Flitsch ◽  
Nicholas J. Turner
Keyword(s):  
Cytochrome P450 ◽  
Cytochromes P450 ◽  
Pcr Primers ◽  
Soluble Form ◽  
Flavin Mononucleotide ◽  
Gene Encoding ◽  
Binding Motifs ◽  
E Coli ◽  
New Class ◽  
C Terminus

ABSTRACT A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

1988 ◽  
Vol 8 (12) ◽  
pp. 5417-5424
Author(s):  
G R Banks ◽  
S Y Taylor
Keyword(s):  
Dna Sequences ◽  
Gene Copy Number ◽  
Mutant Gene ◽  
Ustilago Maydis ◽  
Gene Copy ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Multiple Transcripts ◽  
Different Types

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

scholarly journals Genetic Characterization of Escherichia coli O157 Isolated From Human Stool Specimens in Wassit Province of Iraq

2020 ◽  
Vol 2 (3) ◽  
pp. 1-8
Author(s):  
SabahM Mohammed Alkaby ◽  
Mohammed Jebur
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Coli Strain ◽  
Detection Methods ◽  
Phylogenetic Study ◽  
Gene Encoding ◽  
E Coli ◽  
Stool Specimens ◽  
Human Infections ◽  
Shiga Toxin Genes

The goal of this study was to assess the severity of diarrhea caused by Shiga toxins produced by Escherichia coli ( STEC) isolated from human infections in Wassit province of Iraq. Stool specimens from (161) sporadic cases of diarrheal Patients with the mean age of (20 years), and  range from 1 month to 75 years, were collected in AL-Karamma Teaching Hospital   between March  to July 2019. Then they were processed by culture, microscopic tests and VITEK which were used for the identification as E. coli. PCR was performed for detecting shiga toxin genes in  E. coli isolates, and rfb gene (encoding O-antigene )   of STEC- O157. DNA sequencing was done on some positive isolates. The results of PCR detected stx genes in 19 (12 %) culture isolates of E. coli isolated from human diarrheal specimens. While 9 cases were positive for stx genes and have rfb gene. DNA sequences that depend on the sequence of the vtx2 gene have shown a highly homologous sequencing identity with NCBI-Blast Escherichia coli strain( O157:H7) isolates from humans and animals. The phylogenetic study revealed a clear genetic relationship between human and animal E. coli isolates and then gene sequencing was deposited with accession number into NCBI-Genbank (0MN944014.1). In conclusion, prevalence of E. coli O157 in humans remains undiagnosed because there are no traditional O157 detection methods in all our hospitals. Our study showed that the use of molecular methods in the detection can be used to detect STECs which cannot be detected using routine methods.

scholarly journals Improving Species Identification of Ancient Mammals Based on Next-Generation Sequencing Data

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 509
Author(s):  
Tian Lan ◽  
Yu Lin ◽  
Jacob Njaramba-Ngatia ◽  
Xiao Guo ◽  
Ren Li ◽  
...  
Keyword(s):  
Species Identification ◽  
Ancient Dna ◽  
Dna Sequences ◽  
Next Generation Sequencing Data ◽  
Whole Genome Sequencing Data ◽  
Local Alignment ◽  
Sequencing Data ◽  
Mammal Species ◽  
Query Coverage ◽  
Ancient Remains

The taxonomical identification merely based on morphology is often difficult for ancient remains. Therefore, universal or specific PCR amplification followed by sequencing and BLAST (basic local alignment search tool) search has become the most frequently used genetic-based method for the species identification of biological samples, including ancient remains. However, it is challenging for these methods to process extremely ancient samples with severe DNA fragmentation and contamination. Here, we applied whole-genome sequencing data from 12 ancient samples with ages ranging from 2.7 to 700 kya to compare different mapping algorithms, and tested different reference databases, mapping similarities and query coverage to explore the best method and mapping parameters that can improve the accuracy of ancient mammal species identification. The selected method and parameters were tested using 152 ancient samples, and 150 of the samples were successfully identified. We further screened the BLAST-based mapping results according to the deamination characteristics of ancient DNA to improve the ability of ancient species identification. Our findings demonstrate a marked improvement to the normal procedures used for ancient species identification, which was achieved through defining the mapping and filtering guidelines to identify true ancient DNA sequences. The guidelines summarized in this study could be valuable in archaeology, paleontology, evolution, and forensic science. For the convenience of the scientific community, we wrote a software script with Perl, called AncSid, which is made available on GitHub.

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