RESUMO
Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.
Assuntos
Baculoviridae , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H9N2 , Vírus da Doença de Newcastle , Spodoptera , Animais , Baculoviridae/genética , Células Sf9 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Doença de Newcastle/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Expressão Gênica , Clonagem Molecular/métodos , Vetores Genéticos/genéticaRESUMO
Kainoid natural products are a series of potent ionotropic glutamate receptor agonists produced by a variety of divergent marine micro- and macro-algae. The key biosynthetic step in the construction of the pyrrolidine ring pharmacophore involves a unique branch of non-heme iron α-ketoglutarate dependent dioxygenases (Fe/αKGs) termed the kainoid synthases. These Fe/αKG homologs catalyze a stereoselective C-H abstraction followed by a radical carbon-carbon bond reaction to form the bioactive core on N-prenylated L-glutamic acid substrates. In this article, we describe the expression, purification, and biochemical characterization of four divergent kainoid synthases (DabC, RadC1, DsKabC, GfKabC). Furthermore, we compare and contrast their substrate preferences and product distributions, and provide some preliminary insight into how to repurpose these enzymes for whole cell biocatalysis.
Assuntos
Proteínas Recombinantes , Especificidade por Substrato , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Expressão Gênica , Clonagem Molecular/métodosRESUMO
BACKGROUND: Phalaenopsis bellina, an orchid native to Borneo, is renowned for its unique appearance. It releases distinct fragrances, which have been linked to the presence of terpenoids. However, the identification and study of sesquiterpene synthase in P. bellina remain limited. In this study, we examines the functional characterisation of terpene synthase (TPS) from P. bellina, known as PbTS, through recombinant protein expression and its manifestation in the flower. METHODS AND RESULTS: Gene annotation of PbTS revealed that the inferred peptide sequence of PbTS comprises 1,680 bp nucleotides encoding 559 amino acids with an estimated molecular mass of 65.2 kDa and a pI value of 5.4. A similarity search against GenBank showed that PbTS shares similarities with the previously published partial sequence of P. bellina (ABW98504.1) and Phalaenopsis equestris (XP_020597359.1 and ABW98503.1). Intriguingly, the phylogenetic analysis places the PbTS gene within the TPS-a group. In silico analysis of PbTS demonstrated stable interactions with farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), and geranylgeranyl pyrophosphate (GGPP). To verify this activity, an in vitro enzyme assay was performed on the PbTS recombinant protein, which successfully converted FPP, GPP, and GGPP into acyclic sesquiterpene ß-farnesene, yielding approximately 0.03 mg/L. Expressional analysis revealed that the PbTS transcript was highly expressed in P. bellina, but its level did not correlate with ß-farnesene levels across various flowering time points and stages. CONCLUSION: The insights gained from this study will enhance the understanding of terpenoid production in P. bellina and aid in the discovery of novel fragrance-related genes in other orchid species.
Assuntos
Alquil e Aril Transferases , Flores , Orchidaceae , Filogenia , Sesquiterpenos , Orchidaceae/genética , Orchidaceae/enzimologia , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Sesquiterpenos/metabolismo , Flores/genética , Flores/enzimologia , Sequência de Aminoácidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Clonagem Molecular/métodos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Regulação da Expressão Gênica de PlantasRESUMO
This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.
Assuntos
Clonagem Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Plasmídeos/genética , Clonagem Molecular/métodos , Mutagênese Sítio-Dirigida/métodos , Reação em Cadeia da Polimerase/métodos , DNA/genéticaRESUMO
Genetic modification of plants fundamentally relies upon customized vector designs. The ever-increasing complexity of transgenic constructs has led to increased adoption of modular cloning systems for their ease of use, cost effectiveness, and rapid prototyping. GreenGate is a modular cloning system catered specifically to designing bespoke, single transcriptional unit vectors for plant transformation-which is also its greatest flaw. MultiGreen seeks to address GreenGate's limitations while maintaining the syntax of the original GreenGate kit. The primary limitations MultiGreen addresses are 1) multiplexing in series, 2) multiplexing in parallel, and 3) repeated cycling of transcriptional unit assembly through binary intermediates. MultiGreen efficiently concatenates bespoke transcriptional units using an additional suite of level 1acceptor vectors which serve as an assembly point for individual transcriptional units prior to final, level 2, condensation of multiple transcriptional units. Assembly with MultiGreen level 1 vectors scales at a maximal rate of 2*âlog6nâ+3 days per assembly, where n represents the number of transcriptional units. Further, MultiGreen level 1 acceptor vectors are binary vectors and can be used directly for plant transformation to further maximize prototyping speed. MultiGreen is a 1:1 expansion of the original GreenGate architecture's grammar and has been demonstrated to efficiently assemble plasmids with multiple transcriptional units. MultiGreen has been validated by using a truncated violacein operon from Chromobacterium violaceum in bacteria and by deconstructing the RUBY reporter for in planta functional validation. MultiGreen currently supports many of our in-house multi transcriptional unit assemblies and will be a valuable strategy for more complex cloning projects.
Assuntos
Clonagem Molecular , Vetores Genéticos , Clonagem Molecular/métodos , Vetores Genéticos/genética , Plantas Geneticamente Modificadas/genética , Transformação GenéticaRESUMO
L-arabinose isomerase (L-AI) is a functional enzyme for the isomerizing of D-galactose to produce D-tagatose. In this study, L-AI-C6-encoding gene from the probiotic Lactobacillus fermentum C6 was cloned and expressed in Bacillus subtilis WB600 for investigating enzymatic characteristics and bioconverting D-tagatose by means of whole-cell catalysis. Results showed that the engineered B. subtilis WB600-pMA5-LAI achieved a maximum specific activity of L-AI-C6 (232.65 ± 15.54 U/mg protein) under cultivation in LB medium at 28 °C for 40 h. The recombinant L-AI-C6 was purified, and enzymatic characteristics test showed its optimum reaction temperature and pH at 60 °C and 8.0, respectively. In addition, L-AI-C6 exhibited good stability within the pH range of 5.5-9.0. By using B. subtilis WB600-pMA5-LAI cells as whole-cell catalyst, the highest D-tagatose yield reached 42.91 ± 0.28 % with D-galactose as substrate, which was 2.41 times that of L. fermentum C6 (17.79 ± 0.11 %). This suggested that the cloning and heterologous expression of L-AI-C6 was an effective strategy for improving D-tagatose conversion by whole-cell catalysis. In brief, the present study demonstrated that the reaction temperature, pH, and stability of L-AI-C6 from L. fermentum C6 meet the demands of industrial application, and the constructed B. subtilis WB600-pMA5-LAI shows promising potential for the whole-cell biotransformation of D-tagatose.
Assuntos
Aldose-Cetose Isomerases , Bacillus subtilis , Hexoses , Limosilactobacillus fermentum , Proteínas Recombinantes , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Hexoses/metabolismo , Hexoses/biossíntese , Limosilactobacillus fermentum/enzimologia , Limosilactobacillus fermentum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Clonagem Molecular/métodos , Estabilidade Enzimática , Galactose/metabolismo , CinéticaRESUMO
A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.
Assuntos
Endo-1,4-beta-Xilanases , Estabilidade Enzimática , Xilanos , Xilanos/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/química , Hidrólise , Actinobacteria/enzimologia , Actinobacteria/genética , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Clonagem Molecular/métodos , Especificidade por Substrato , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Pichia/genética , Pichia/metabolismo , Actinomycetales/enzimologia , Actinomycetales/genética , Sequência de Aminoácidos , SaccharomycetalesRESUMO
Pacific abalone (Haliotis discus hannai) is a marine gastropod mollusc with significant economic importance in both global fisheries and aquaculture. However, studies exploring the gonadal development and regulatory mechanisms of Haliotis discus hannai are limited. This study aimed to explore whether the vasa gene acted as a molecular marker for germ cells. Initially, the vasa gene was successfully cloned using the cDNA-end rapid amplification technique. The cloned gene had a 2478-bp-long open reading frame and encoded 825 amino acids. Then, a recombinant expression vector was constructed based on the Vasa protein, and an 87-kDa recombinant protein was prepared. Subsequently, a polyclonal antibody was prepared using the purified recombinant protein. The enzyme-linked immunosorbent assay (ELISA) confirmed the titer of the antibody to be ≥512 K. The immunohistochemical analysis revealed that Vasa was widely expressed in oogonia, Stage I oocytes, spermatogonia, and primary spermatocytes. The specific expression of Vasa in the hermaphroditic gonads of abalone was assessed using western blotting to investigate the effects of different photoperiods (12 L:12D, 24 L:0D, 18 L:6D, and 6 L:18D) on the gonadal development of abalone (P < 0.05), with higher expression levels observed in the ovarian proliferative and spermary maturing stages compared with other developmental stages (P < 0.05). Additionally, Vasa exhibited the highest expression in the spermary and ovary under a photoperiod of 18 L:6D (P < 0.05). These data demonstrated the key role of Vasa in developing germ cells in abalone. They shed light upon the molecular mechanism through which the photoperiod influenced Vasa expression and regulated gonadal development in abalone. The findings might provide theoretical references for analyzing the differentiation pattern of abalone germ cells and the genetic improvement and conservation of germplasm resources.
Assuntos
RNA Helicases DEAD-box , Gastrópodes , Animais , Feminino , Masculino , Sequência de Aminoácidos , Clonagem Molecular/métodos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Gametogênese/genética , Gastrópodes/genética , Gônadas/metabolismo , FotoperíodoRESUMO
Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistance to abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elongase 1 (FAE1) gene encodes the enzyme ß-ketoacyl CoA synthase (KCS), a crucial enzyme in the biosynthesis of erucic acid. In this study, the isolation and cloning of the FAE1 gene from Camelina sativa were conducted to construct an antisense structure. The molecular homology modeling of DFAE1 proteins using the SWISS-MODEL server on ExPASy led to the generation of the 3D structures of FAE1 and DFAE1 proteins. The GMQE values of 0.44 for FAE1 and 0.08 for DFAE1 suggest high accuracy in the structural estimation of these genes. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning the FAE1 gene into the Bluescript II SK+ vector and sequencing, the resulting fragments were utilized to construct the antisense structure in the pBI121 plant expression vector. The approved antisense structure was introduced into the Camelina plant using the Agrobacterium-mediated method, with optimization of tissue culture and gene transfer conditions. This approach holds potential to advance our knowledge of fat biosynthesis, leading to potential improvements in oil quality in Camelina sativa.
Assuntos
Brassicaceae , Clonagem Molecular , Ácidos Erúcicos , Elongases de Ácidos Graxos , Brassicaceae/genética , Brassicaceae/metabolismo , Clonagem Molecular/métodos , Ácidos Erúcicos/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Sementes/genética , Sementes/metabolismo , Modelos Moleculares , Regulação da Expressão Gênica de Plantas , Acetiltransferases/genética , Acetiltransferases/metabolismo , Genes de PlantasRESUMO
Improving crop plants using biotechnological implications is a promising and modern approach compared to traditional methods. High-temperature exposure to the reproductive stage induces flower abortion and declines grain filling performance, leading to smaller grain production and low yield in lentil and other legumes. Thus, cloning effective candidate genes and their implication in temperature stress tolerance in lentil (Lens culinaris Medik.) using biotechnological tools is highly demandable. The 12-oxophytodienoic acid reductases (OPRs) are flavin mononucleotide-dependent oxidoreductases with vital roles in plants. They are members of the old yellow enzyme (OYE) family. These enzymes are involved in the octadecanoid pathway, which contributes to jasmonic acid biosynthesis and is essential in plant stress responses. Lentil is one of the vital legume crops affected by the temperature fluctuations caused by global warming. Therefore, in this study, the LcOPR1 gene was successfully cloned and isolated from lentils using RT-PCR to evaluate its functional responses in lentil under heat stress. The bioinformatics analysis revealed that the full-length cDNA of LcOPR1 was 1303 bp, containing an 1134 bp open reading frames (ORFs), encoding 377 amino acids with a predicted molecular weight of 41.63 and a theoretical isoelectric point of 5.61. Bioinformatics analyses revealed that the deduced LcOPR1 possesses considerable homology with other plant 12-oxophytodienoic acid reductases (OPRs). Phylogenetic tree analysis showed that LcOPR1 has an evolutionary relationship with other OPRs in different plant species of subgroup I, containing enzymes that are not required for jasmonic acid biosynthesis. The expression analysis of LcOPR1 indicated that this gene is upregulated in response to the heat-stress condition and during recovery in lentil. This study finding might be helpful to plant breeders and biotechnologists in LcOPR1 engineering and/or plant breeding programs in revealing the biological functions of LcOPR1 in lentils and the possibility of enhancing heat stress tolerance by overexpressing LcOPR1 in lentil and other legume plants under high temperature.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Lens (Planta) , Filogenia , Lens (Planta)/genética , Lens (Planta)/enzimologia , Clonagem Molecular/métodos , Regulação da Expressão Gênica de Plantas/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Sequência de Aminoácidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Temperatura Alta , Genes de Plantas , Resposta ao Choque Térmico/genética , Oxilipinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CHRESUMO
In this study, we describe a novel method for one-step cloning and targeted duplication of P. ananatis chromosomal fragments. According to this method, the chromosomal region of interest is subcloned in vivo via λ Red recombination into the short synthetic non-replicable DNA fragment containing the excisable antibiotic-resistance marker gene and φ80 att-P site. The resulting circular non-replicating DNA molecule was immediately inserted into an alternative chromosomal locus due to φ80-integrase activity. To this end, the specially designed helper plasmid pONI, which can provide both the λ Red recombineering and φ80-integrase-mediated insertion, was constructed. In the described method, PCR amplification of the cloning fragment is unnecessary, making it convenient for manipulation of long-length DNA. Additionally, the possibility of spontaneous mutations occurring is completely precluded. This method was effectively used for the targeted chromosomal integration of additional copies of individual genes and operons up to 16 kb in size.
Assuntos
Cromossomos Bacterianos , Clonagem Molecular , Pantoea , Plasmídeos , Pantoea/genética , Clonagem Molecular/métodos , Cromossomos Bacterianos/genética , Plasmídeos/genética , DNA Bacteriano/genética , Recombinação Genética , Integrases/genéticaRESUMO
We have created a novel synthetic biology expression system allowing easy refactoring of biosynthetic gene clusters (BGCs) as monocistronic transcriptional units. The system is based on a set of plasmids containing a strong kasOp* promoter, RBS and terminators. It allows the cloning of biosynthetic genes into transcriptional units kasOp*-gene(s)-terminator flanked by several rare restriction cloning sites that can be sequentially combined into the artificial BGC in three compatible Streptomyces integration vectors. They allow a simultaneous integration of these BGCs at three different attB sites in the Streptomyces chromosome. The system was validated with biosynthetic genes from two known BGCs for aromatic polyketides landomycin and mithramycin.
Assuntos
Antibacterianos , Família Multigênica , Streptomyces , Biologia Sintética , Biologia Sintética/métodos , Antibacterianos/biossíntese , Antibacterianos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Família Multigênica/genética , Plasmídeos/genética , Metabolismo Secundário/genética , Regiões Promotoras Genéticas/genética , Clonagem Molecular/métodosRESUMO
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.
Assuntos
Baculoviridae , Proteínas Recombinantes , Vírus da Febre do Vale do Rift , Proteínas do Envelope Viral , Baculoviridae/genética , Animais , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus da Febre do Vale do Rift/genética , Células Sf9 , Expressão Gênica , Humanos , Vetores Genéticos/genética , Clonagem Molecular/métodosRESUMO
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.
Assuntos
Mapeamento Cromossômico , Clonagem Molecular , Hordeum , Dormência de Plantas , Locos de Características Quantitativas , Hordeum/genética , Hordeum/crescimento & desenvolvimento , Dormência de Plantas/genética , Mapeamento Cromossômico/métodos , Clonagem Molecular/métodos , Genes de Plantas , Sementes/genética , Sementes/crescimento & desenvolvimento , Cromossomos de Plantas/genéticaRESUMO
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: â¢The in vivo cloning was performed by replacing the target gene with the landing pad. â¢Fast eradication of non-recombinant plasmids was possible by adapting key vectors. â¢This strategy is not dependent on in vitro assembly reactions and expensive materials.
Assuntos
Clonagem Molecular , Escherichia coli , Plasmídeos , Reação em Cadeia da Polimerase , Recombinação Genética , Escherichia coli/genética , Clonagem Molecular/métodos , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Vetores Genéticos/genética , Sistemas CRISPR-CasRESUMO
Synthetic promoters are powerful tools to boost the biotechnological potential of microalgae as eco-sustainable industrial hosts. The increasing availability of transcriptome data on microalgae in a variety of environmental conditions allows to identify cis-regulatory elements (CREs) that are responsible for the transcriptional output. Furthermore, advanced cloning technologies, such as golden gate-based MoClo toolkits, enable the creation of modular constructs for testing multiple promoters and a range of reporter systems in a convenient manner. In this chapter, we will describe how to introduce in silico-identified CREs into promoter sequences, and how to clone the modified promoters into MoClo compatible vectors. We will then describe how these promoters can be evaluated and screened for transgene expression in an established microalgal model for genetic perturbation, i.e., Chlamydomonas reinhardtii.
Assuntos
Chlamydomonas reinhardtii , Regiões Promotoras Genéticas , Chlamydomonas reinhardtii/genética , Vetores Genéticos/genética , Clonagem Molecular/métodos , Transgenes , Biologia Sintética/métodos , Microalgas/genética , Engenharia Genética/métodosRESUMO
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).
Assuntos
DNA Ribossômico , Naegleria , Transfecção , Naegleria/genética , Transfecção/métodos , DNA Ribossômico/genética , Trofozoítos , DNA de Protozoário/genética , Clonagem Molecular/métodosRESUMO
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.
Assuntos
Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Expressão Gênica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Cromatografia de Afinidade , Histidina/metabolismo , Histidina/genética , Plasmídeos/genética , Clonagem Molecular/métodos , Cromatografia em Gel , OligopeptídeosRESUMO
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.
Assuntos
Clonagem Molecular , Plasmídeos , Plasmídeos/genética , Clonagem Molecular/métodos , Vetores Genéticos/genética , Baculoviridae/genética , Cromossomos Artificiais Bacterianos/genéticaRESUMO
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.