In vivo cloning of PCR product via site-specific recombination in Escherichia coli.

Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

Cloning and characterization of the histone variant gene H2A.Z in Bombyx mori.

H2A.Z, the most evolutionarily conserved variant of histone H2A, plays a pivotal role in chromatin remodeling and contributes significantly to gene transcription and genome stability. However, the role of H2A.Z in the silkworm (Bombyx mori) remains unclear. In this study, we cloned the BmH2A.Z from B. mori. The open reading frame of BmH2A.Z is 390 bp, encoding 129 amino acids, with a confirmed molecular weight of 13.4 kDa through prokaryotic expression analysis. Sequence analysis revealed that BmH2A.Z has a conserved H2A.Z domain and is closely related to the systemic evolution of other known H2A.Zs. The expression profile of BmH2A.Z at various developmental stages of the B. mori exhibited the highest expression level in the 1st instar, followed by the grain stage and the 2nd instar, and the lowest expression level in the moth. The highest transcript level of BmH2A.Z was observed in the head, with relatively lower levels detected in the blood than in the other tissues under consideration. In addition, the upregulation of BmH2A.Z resulted in the amplified expression of B. mori nucleopolyhedrovirus (BmNPV) genes, thus facilitating the proliferation of BmNPV. This study establishes a foundation for investigating the role of BmH2A.Z in B. mori and its participation in virus-host interactions.

[Cloning and functional characterization of the pinoresinol-lariciresinol reductase gene IiPLR2 in Isatis indigotica].

The pinoresinol-lariciresinol reductase (PLR), a crucial enzyme in the biosynthesis of lignans in plants, catalyzes a two-step reaction to produce lariciresinol and secoisolariciresinol. Lignans such as lariciresinol are the effective components of traditional Chinese medicine Radix Isatidis in exerting antiviral activity. In order to study the function of the key enzyme PLR in the biosynthesis of lariciresinol in Isatis indigotica, the original plant of Radix Isatidis, IiPLR2 was cloned from I. indigotica, with a full length of 954 bp, encoding 317 amino acids. Multiple sequence alignment showed that IiPLR2 contained a conserved nicotinamide adenine dinucleotide phosphate (NADPH)-binding motif. The phylogenetic tree showcased that IiPLR2 shared the same clade with AtPrR1 from Arabidopsis thaliana. The prokaryotic expression vector pET32a-IiPLR2 was constructed and then transformed into Escherichia coli BL21(DE3) competent cells for protein expression. The purified enzyme IiPLR2 could catalyze the conversion of pinoresinol to lariciresinol and the conversion of lariciresinol to secoisolariciresinol. The cloning, sequencing, and catalytic functional analysis of IiPLR2 in this study enrich the understanding of this kind of functional proteins in I. indigotica and supplement the biosynthesis pathways of lignans. Moreover, this study provides a functional module for further research on metabolic regulation and synthetic biology and lays a foundation for comprehensively revealing the relationship between the spatial structures and catalytic functions of such proteins.

[An in vitro self-ligation-based method for constructing infectious DNA clone of porcine circovirus type 2].

The aim of this study was to establish a rapid method for constructing infectious clones of porcine circovirus type 2 (PCV2). In this study, we constructed circular infectious clones of PCV2 by seamless cloning technology, using the clinically isolated strain PCV2-LX as a template. Meanwhile, this method was compared with the conventional restriction-ligation approach, focusing on the in vitro circularization (self-ligation) process of the genome and the growth characteristics of rescued viruses. The results showed that this method eliminates the need to analyze and introduce restriction endonuclease sites, thus avoiding the complexities associated with traditional restriction enzyme-based cloning steps. It offers a simple and rapid operation, enabling more efficient editing of the PCV2 genome. The infectious clones constructed using this method could be successfully rescued through liposome transfection, resulting in the production of recombinant viruses that could be stably passaged. Moreover, the recombinant viruses rescued by this method exhibited enhanced proliferative capacity in PK-15 cells and 3D4/31 cells (immortalized porcine alveolar macrophages). In conclusion, this study has established a novel reverse genetics system for PCV2, providing a new strategy for the development of PCV2 genetic engineering vaccines. Additionally, it serves as a reference for the construction of infectious clones for other emerging circoviruses such as PCV3 and PCV4.

Heterologous Expression and Purification of Eukaryotic ALA Synthase from E. coli.

This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms.

The Discovery, Molecular Cloning, and Characterization of Dextransucrase LmDexA and Its Active Truncated Mutant from Leuconostoc mesenteroides NN710.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, LmDexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190LmDexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190LmDexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a Km of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190LmDexA. ΔN190LmDexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

TOPO Cloning for Efficient Plasmid Construction.

This chapter outlines the use of TOPO cloning for streamlined generation of a recombinant plasmid containing your gene of interest for use in the Bac-to-Bac™ Baculovirus Expression System.

Heterologous expression of a novel galactose-1-phosphate uridylyltransferase from Thermodesulfatator indicus and its application for bioproduction of Gal-ß-1,4-GlcNAc-X.

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and ß-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-ß-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-ß-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Modular Assembly of Synthetic Secondary Chromosomes.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.

Map-Based Cloning of the Causal Gene for a Seed Dormancy Quantitative Trait Locus in Barley.

Seed dormancy is an important agronomic trait in cereal crops. Throughout the domestication of cereals, seed dormancy has been reduced to obtain uniform germination. However, grain crops must retain moderate levels of seed dormancy to prevent problems such as preharvest sprouting in wheat (Triticum aestivum) and barley (Hordeum vulgare). To produce modern cultivars with the appropriate seed dormancy levels, it is important to identify the genes responsible for seed dormancy. With recent advances in sequencing technology, several causal genes for seed dormancy quantitative trait loci (QTLs) have been identified in barley and wheat. Here, we present a method to identify causal genes for seed dormancy QTLs in barley, a method that is also applicable to other cereals.

Optimization, gene cloning, expression, and molecular docking insights for enhanced cellulase enzyme production by Bacillus amyloliquefaciens strain elh1.

BACKGROUND: In this study, we isolated a cellulase-producing bacterium, Bacillus amyloliquefaciens strain elh, from rice peel. We employed two optimization methods to enhance the yield of cellulase. Firstly, we utilized a one-variable-at-a-time (OVAT) approach to evaluate the impact of individual physical and chemical parameters. Subsequently, we employed response surface methodology (RSM) to investigate the interactions among these factors. We heterologously expressed the cellulase encoding gene using a cloning vectorin E. coli DH5α. Moreover, we conducted in silico molecular docking analysis to analyze the interaction between cellulase and carboxymethyl cellulose as a substrate. RESULTS: The bacterial isolate eh1 exhibited an initial cellulase activity of 0.141 ± 0.077 U/ml when cultured in a specific medium, namely Basic Liquid Media (BLM), with rice peel as a substrate. This strain was identified as Bacillus amyloliquefaciens strain elh1 through 16S rRNA sequencing, assigned the accession number OR920278 in GenBank. The optimal incubation time was found to be 72 h of fermentation. Urea was identified as the most suitable nitrogen source, and dextrose as the optimal sugar, resulting in a production increase to 5.04 ± 0.120 U/ml. The peak activity of cellulase reached 14.04 ± 0.42 U/ml utilizing statistical optimization using Response Surface Methodology (RSM). This process comprised an initial screening utilizing the Plackett-Burman design and further refinement employing the BOX -Behnken Design. The gene responsible for cellulase production, egl, was effectively cloned and expressed in E. coli DH5α. The transformed cells exhibited a cellulase activity of 22.3 ± 0.24 U/ml. The egl gene sequence was deposited in GenBank with the accession number PP194445. In silico molecular docking revealed that the two hydroxyl groups of carboxymethyl cellulose bind to the residues of Glu169 inside the binding pocket of the CMCase. This interaction forms two hydrogen bonds, with an affinity score of -5.71. CONCLUSIONS: Optimization of cultural conditions significantly enhances the yield of cellulase enzyme when compared to unoptimized culturing conditions. Additionally, heterologous expression of egl gene showed that the recombinant form of the cellulase is active and that a valid expression system can contribute to a better yield of the enzyme.

Expression and Functional Analysis of the Metallothionein and Metal-Responsive Transcription Factor 1 in Phascolosoma esculenta under Zn Stress.

Metallothioneins (MTs) are non-enzymatic metal-binding proteins widely found in animals, plants, and microorganisms and are regulated by metal-responsive transcription factor 1 (MTF1). MT and MTF1 play crucial roles in detoxification, antioxidation, and anti-apoptosis. Therefore, they are key factors allowing organisms to endure the toxicity of heavy metal pollution. Phascolosoma esculenta is a marine invertebrate that inhabits intertidal zones and has a high tolerance to heavy metal stress. In this study, we cloned and identified MT and MTF1 genes from P. esculenta (designated as PeMT and PeMTF1). PeMT and PeMTF1 were widely expressed in all tissues and highly expressed in the intestine. When exposed to 16.8, 33.6, and 84 mg/L of zinc ions, the expression levels of PeMT and PeMTF1 in the intestine increased first and then decreased, peaking at 12 and 6 h, respectively, indicating that both PeMT and PeMTF1 rapidly responded to Zn stress. The recombinant pGEX-6p-1-MT protein enhanced the Zn tolerance of Escherichia coli and showed a dose-dependent ABTS free radical scavenging ability. After RNA interference (RNAi) with PeMT and 24 h of Zn stress, the oxidative stress indices (MDA content, SOD activity, and GSH content) and the apoptosis indices (Caspase 3, Caspase 8, and Caspase 9 activities) were significantly increased, implying that PeMT plays an important role in Zn detoxification, antioxidation, and anti-apoptosis. Moreover, the expression level of PeMT in the intestine was significantly decreased after RNAi with PeMTF1 and 24 h of Zn stress, which preliminarily proved that PeMTF1 has a regulatory effect on PeMT. Our data suggest that PeMT and PeMTF1 play important roles in the resistance of P. esculenta to Zn stress and are the key factors allowing P. esculenta to endure the toxicity of Zn.

Expression and Purification of RVFV Glycoprotein C for Structural Studies Using Baculovirus Expression System.

The Rift Valley fever virus (RVFV), transmitted through mosquito bites, leads to severe illness in humans and livestock throughout Africa and the Arabian Peninsula, causing significant morbidity and mortality. As of now, there are no verified and efficacious drugs or licensed vaccines accessible for the prevention or treatment of RVFV infections in both humans and livestock. The mature RVFV virion has two envelope proteins on its surface: glycoprotein N (GN) and glycoprotein C (GC). These proteins play a significant role in facilitating the virus's entry into the host cell, making them prominent targets for entry mechanism research as well as targets for drugs and vaccine development. The initial stage in obtaining atomic-resolution structural and mechanistic information on viral entry as well as developing biochemical and biophysical research tools involves recombinant protein production. In this chapter, we describe a simplified and scalable protocol facilitating the generation of high-quality, high-titer baculovirus virus for expression and purification of RVFV GC, utilizing the baculovirus-mediated expression system in insect cells.

[Cloning and functional characterization of oxidosqualene cyclase gene for α-amyrin synthesis].

Ursolic acid has gradually attracted much attention due to its unique pharmacological activities and valuable market value in recent years. Currently, ursolic acid is mostly extracted from loquat leaves, but the plant extraction method has low yield and high cost, and chemical synthesis is not readily available, so the biosynthesis method provides a new source for ursolic acid. α-amyrin acts as the main precursor for the synthesis of ursolic acid, and its yield is positively correlated with ursolic acid yield. Oxidosqualene cyclase(OSC) belongs to a multigene family which can catalyze the common precursor 2,3-oxidosqualene to generate different types of triterpene backbones, and plays a decisive role in the synthesis of triterpenoids. However, there are fewer reported key genes catalyzing the synthesis of α-amyrin in medicinal plants, and the yield and proportion of α-amyrin in the catalyzed products have always been a focus of research. In this study, ItOSC2, MdOSC1, AaOSC2 and CrAS, four enzymes capable of catalyzing the production of α-amyrin from 2,3-oxidosqualene, were cloned from Iris tectorum, Malus domestica, Artemisia annua and Catharanthus roseus, subject to sequence alignment and phylogenetic tree analyses, and transformed into Saccharomyces cerevisiae as plasmids. After 7 days of fermentation, the yield and proportions of α-amyrin, ß-amyrin and ergosterol were measured. Finally, AaOSC2 with the best ability to catalyze the generation of α-amyrin was filtered out, providing a key gene element for the later construction of engineered yeast strains with high production of α-amyrin and ursolic acid.

[Gene cloning and functional analysis of an iridoid oxidase gene in Rehmannia glutinosa].

Rehmannia glutinosa is one of the commonly used Chinese herbal medicines, which has activities of heat-clearing,blood-cooling, Yin-nourishing, and body fluid-promoting. Iridoid glycosides are the main bioactive in R. glutinosa. Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides. In this study, an iridoid oxidase gene Rg IO was screened based on the transcriptome data, followed by bioinformatics analysis, expression characteristic detection, and subcellular localization analysis. The results show that the coding region of Rg IO is 1 536 bp, with 511 amino acids encoded, and the molecular weight is about 58 258. 01. The protein sequence of Rg IO contains the conserved domains and motifs of cytochrome P450 oxidases. Rg IO has the highest sequence identities with its ortholog proteins in Striga asiatica, Striga hermonthica, and Centranthera grandiflora and has good sequence identities(77. 28%) with Catharanthus roseus Cr IO. Rg IO shows specific expression in the leaf of R. glutinosa. In response to MeJA induction, the expression of MeJA in leaves and roots after treatment increases by 3. 15 and 1. 3 times at 3 h and 6 h,respectively. The result of subcellular localization shows that Rg IO is distributed in the endoplasmic reticulum. Agrobacterium-mediated transient expression of Rg IO gene in leaves of R. glutinosa makes the content of catalpol increase by 0. 82 times compared with the transient expression of the empty vector. This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R. glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.

Molecular characterization of EcCLP1, a new putative cathepsin L protease from Echinococcus canadensis.

Echinococcus granulosus sensu lato is a platyhelminth parasite and the etiological cause of cystic echinococcosis (CE), a zoonotic and neglected disease that infects animals and humans worldwide. As a part of the biological arsenal of the parasite, cathepsin L proteases are a group of proteins that are believed to be essential for parasite penetration, immune evasion, and establishment in the tissues of the host. In this work, we have cloned and sequenced a new putative cathepsin L protease from Echinococcus canadensis (EcCLP1). The bioinformatic analysis suggests that EcCLP1 could be synthesized as a zymogen and activated after proteolytic cleavage. The multiple sequence alignment with other cathepsin proteases reveals important functional conserved features like a conserved active site, an N-linked glycosylation residue, a catalytic triad, an oxyanion hole, and three putative disulfide bonds. The phylogenetic analysis suggests that EcCLP1 could indeed be a cathepsin L cysteine protease from clade 1 as it grouped with cathepsins from other species in this clade. Modeling studies suggest that EcCLP1 has two domains forming a cleft where the active site is located and an occluding role for the propeptide. The transcriptomic analysis reveals different levels of cathepsin transcript expression along the different stages of the parasite life cycle. The whole-mount immunohistochemistry shows an interesting superficial punctate pattern of staining which suggests a secretory pattern of expression. The putative cathepsin L protease characterized here may represent an interesting tool for diagnostic purposes, vaccine design, or a new pharmacological target for antiparasitic intervention.

Title: Caractérisation moléculaire d'EcCLP1, une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis. Abstract: Echinococcus granulosus sensu lato est un Plathelminthe parasite et la cause étiologique de l'échinococcose kystique (EK), une maladie zoonotique et négligée qui infecte les animaux et les humains dans le monde entier. En tant que partie de l'arsenal biologique du parasite, les protéases de type cathepsine L sont un groupe de protéines considérées comme essentielles à la pénétration du parasite, l'évasion immunitaire et son établissement dans les tissus de l'hôte. Dans ce travail, nous avons cloné et séquencé une nouvelle protéase putative de type cathepsine L d'Echinococcus canadensis (EcCLP1). L'analyse bioinformatique suggère qu'EcCLP1 pourrait être synthétisée sous forme de zymogène et activée après clivage protéolytique. L'alignement de séquences multiples avec d'autres protéases de type cathepsine révèle d'importantes caractéristiques fonctionnelles conservées telles qu'un site actif conservé, un résidu de glycosylation lié à N, une triade catalytique, un trou oxyanion et trois liaisons disulfure putatives. L'analyse phylogénétique suggère qu'EcCLP1 pourrait en effet être une protéase de type cathepsine L du clade 1 car elle se regroupe avec les cathepsines d'autres espèces de ce clade. Les études de modélisation suggèrent qu'EcCLP1 possède deux domaines formant une fente où se trouve le site actif et un rôle d'occlusion pour le propeptide. L'analyse transcriptomique révèle différents niveaux d'expression du transcrit de la cathepsine au cours des différentes étapes du cycle de vie du parasite. L'immunohistochimie de montages entiers montre un intéressant motif de coloration ponctuée superficielle qui suggère un modèle d'expression sécrétoire. La protéase putative de type cathepsine L caractérisée ici peut représenter un outil intéressant à des fins de diagnostic, de conception de vaccins ou une nouvelle cible pharmacologique pour une intervention antiparasitaire.

Optimizing CaCl2-mediated transformation of Pseudomonas aeruginosa SDK-6 with pJN105 using OFAT: A novel and efficient cloning approach.

Cloning and expression of a gene in the desired host is required for optimum production in recombinant strains. The present research is the first attempt to optimize the physiological conditions for the transformation of Pseudomonas aeruginosa SDK-6 with pJN105. Different factors, such as inoculum size, incubation period, heat shock temperature, and heat shock time were optimized using one factor at a time (OFAT) followed by the selection of transformants using gentamicin resistance marker. The maximum number of transformants (2.002 ± 0.077 × 105 cfu/ µg of plasmid DNA) were reported with 0.5% (v/v) inoculum, an incubation period of 3 h, and heat shock treatment at 50 °C for 1 min. An overall 12-fold increase in transformation efficiency was observed. The presence of a 6055 bp band on agarose gel confirmed the transformation of Pseudomonas aeruginosa with the vector pJN105.

Cloning and functional analysis of the juvenile hormone receptor gene CsMet in Coccinella septempunctata.

The potential role of the juvenile hormone receptor gene (methoprene-tolerant, Met) in reproduction of Coccinella septempunctata L. (Coleoptera: Coccinellidae)(Coleoptera: Coccinellidae), was investigated by cloning, analyzing expression profiles by quantitative real-time PCR, and via RNA interference (RNAi). CsMet encoded a 1518-bp open reading frames with a predicted protein product of 505 amino acids; the latter contained 2 Per-Arnt-Sim repeat profile at amino acid residues 30-83 and 102-175. CsMet was expressed in different C. septempunctata larvae developmental stages and was most highly expressed in third instar. CsMet expression in female adults gradually increased from 20 to 30 d, and expression levels at 25 and 30 d were significantly higher than levels at 1-15 d. CsMet expression in 20-d-old male adults was significantly higher than in males aged 1-15 d. CsMet expression levels in fat body tissues of male and female adults were significantly higher than expression in the head, thorax, and reproductive system. At 5 and 10 d after CsMet-dsRNA injection, CsMet expression was significantly lower than the controls by 75.05% and 58.38%, respectively. Ovary development and vitellogenesis in C. septempunctata injected with CsMet-dsRNA were significantly delayed and fewer mature eggs were produced. This study provides valuable information for the large-scale rearing of C. septempunctata.