Gateway Cloning Strategy Research Articles

Mobilization of Plasmids From Bacteria Into Diatoms by Conjugation Technique.

Diatoms serve as a source for a variety of compounds with particular biotechnological interest. Therefore, redirecting the flow to a specific pathway requires the elucidation of the gene's specific function. The most commonly used method in diatoms is biolistic transformation, which is a very expensive and time-consuming method. The use of episomes that are maintained as closed circles at a copy number equivalent to native chromosomes has become a useful genetic system for protein expression that avoids multiple insertions, position-specific effects on expression, and potential knockout of non-targeted genes. These episomes can be introduced from bacteria into diatoms via conjugation. Here, we describe a detailed protocol for gene expression that includes 1) the gateway cloning strategy and 2) the conjugation protocol for the mobilization of plasmids from bacteria to diatoms.

Genetic manipulation of pea (Pisum sativum L.) with Arabidopsisˈs heat shock factor HsfA1d improves ROS scavenging system to confront thermal stress

Arabidopsisˈs HsfA1d was found to confer resistance to pea plant against heat stress by enhancing the activity of antioxidant enzymes and decreasing hydrogen per oxide compared to wild type plants. Pea is one of the important cool season food crops. Heat is the most critical abiotic stress that adversely affects pea growth and economic yield. Response to heat stress in plants is mostly regulated by heat shock factors (HSFs). The current study aimed at improving heat tolerance of pea plant by incorporating heat shock factor, HsfA1d, isolated from Arabidopsis thaliana. Gateway cloning strategy was used for cloning of HsfA1d in plant expression vector pGWB415. The target gene was introduced into pea plant using Agrobacterium-mediated transformation approach. Transformant pea plants revealed five fold increase in the expression of HsfA1d under heat stress (42 °C) compared to room temperature (25 °C). Transgenic and wild type plants exhibited significant difference in their H2O2 content, under thermal stress. Upon exposure to heat stress, wild type plants shown significant increase in their H2O2 content while no such elevation was found in transgenic plants. Similarly, significant difference in superoxide dismutase activities of transgenic and wild type plants was recorded, under thermal stress. Heat stress induced more pronounced increase in SOD activities of transgenic plants compared to wild type. Highly significant increase in proline content and ascorbate peroxidase activities of transgenic plants was observed upon exposure to thermal stress.

Introduction of Arabidopsis's heat shock factor HsfA1d mitigates adverse effects of heat stress on potato (Solanum tuberosum L.) plant.

Thermal stress induces a wide array of morphological and physiological changes in potato affecting its development and economic yield. Response to thermal stress in plants is mostly regulated by heat shock factors (hsfs). The current study aimed at improving heat tolerance by transforming potato plant with heat shock factor, HsfA1d, using Agrobacterium. Gateway cloning strategy was adopted for isolation of HsfA1d from Arabidopsis thaliana and cloning into plant expression vector. The target gene was introduced into potato by infecting internodal explants with Agrobacterium strain GV3101 carrying pGWB402Ω-HsfA1d construct. Upon exposure to heat stress, the wild-type plants turned yellowish, whereas no phenotypic effect on transgenic plants was observed. Expression of HsfA1d in transgenic plants was increased by 5.8-fold under thermal stress compared to room temperature. Transgenic plants exhibited 6-fold increase in the expression of downstream HSP70 under thermal stress compared to wild-type plants. Both chlorophyll a and b were significantly decreased in wild-type plants while no such decrease was recorded in transgenic plants under thermal stress. Heat stress was found to have no significant effect on carotenoid pigments of both wild-type and transgenic plants. Significantly lower electrolyte leakage from transgenic plants was witnessed compared to wild type upon exposure to thermal stress. Transgenic plants accumulated significantly higher proline content compared to wild-type plants under heat stress. It is concluded that HsfA1d plays a vital role in plant thermotolerance and hence can be effectively used to enhance the resistance of crop plants against heat stress.

Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.

Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.

Simultaneous Expression of AhBTF3, AhNF-YA7 and EcZF Modulates Acclimation Responses to Abiotic Stresses in Rice (Oryza sativa L)

IntroductionAbiotic stress responses are under the strict control of transcriptional regulation. Gene stacking approach using two to three transcription factors (TFs), which aid in stable transcription would be promising. An attempt was made to co express a basal transcription factor (AhBTF3); Nuclear factor (AhNFYA7), an activator protein and zinc finger protein (EcZF), a stress inducible TF. It is hypothesised that a combination of different classes of TFs enhances transcriptional stability that can regulate stress acclimation. MethodsThe gene cassette harbouring NFYA7, BTF3 and ZF expressed under rbcS, Ubi and rd29A promoters respectively, was cloned in the destination vector pi12GW (PsAKR1 as selectable marker), by modified gateway cloning strategy. Agrobacterium mediated in planta transformation technique was used to generate rice (AC-39020) transgenic plants. The putative transgenics were screened for glyphosate tolerance and confirmed by PCR and RT-PCR. The select lines were subjected for physiological characterisation to assess their stress response. ResultsThe transformants screened for glyphosate tolerance exhibited varied response (Fig. 1). The select lines maintained higher chlorophyll stability when subjected for excised leaf disc stress tolerance assay using PEG and NaCl (Fig. 2A and B). The transgenics exhibited better growth as evidenced by plant height and seed yield (Fig. 2C and D).The promising transgenic lines were challenged with different abiotic stresses using NaCl, PEG and cadmium chloride which revealed their superior performance at seedling level (Fig. 3A). The integration of marker gene and expression of each transgene was confirmed by PCR (Fig. 3B) and RT-PCR respectively (Fig. 3C). DiscussionCo expression of stress relevant TFs can enhance the levels of tolerance tomultiple abiotic stresses as demonstrated in this study. The roles of individual TF in modulating cellular stress responses need to be examined.

Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis

Stress adaptation in plants involves altered expression of many genes through complex signaling pathways. To achieve the optimum expression of downstream functional genes, we expressed AtbHLH17 (AtAIB) and AtWRKY28 TFs which are known to be upregulated under drought and oxidative stress, respectively in Arabidopsis. Multigene expression cassette with these two TFs and reporter gene GUS was developed using modified gateway cloning strategy. The GUS assay and expression analysis of transgenes in transgenic plants confirmed the integration of multigene cassette. The transgenic lines exhibited enhanced tolerance to NaCl, Mannitol and oxidative stress. Under mannitol stress condition significantly higher root growth was observed in transgenics. Growth under stress and recovery growth was substantially superior in transgenics exposed to gradual long term desiccation stress conditions. We demonstrate the expression of several downstream target genes under various stress conditions. A few genes having either WRKY or bHLH cis elements in their promoter regions showed higher transcript levels than wild type. However, the genes which did not have either of the motifs did not differ in their expression levels in stress conditions compared to wild type plants. Hence co-expressing two or more TFs may result in upregulation of many downstream target genes and substantially improve the stress tolerance of the plants.

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.

WITHDRAWN: Reprint of: Effective high-throughput overproduction of membrane proteins in Escherichia coli

Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-β-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2mg/l, with 18 targets producing at levels of 5mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.

Host-derived suppression of nematode reproductive and fitness genes decreases fecundity of Heterodera glycines Ichinohe

To control Heterodera glycines Ichinohe (soybean cyst nematode) in Glycine max (L.) Merr. (soybean), we evaluated the use of producing transgenic soybean seedlings expressing small interfering RNAs (siRNAs) against specific H. glycines genes. Gene fragments of three genes related to nematode reproduction or fitness (Cpn-1, Y25 and Prp-17) were PCR-amplified using specific primers and independently cloned into the pANDA35HK RNAi vector using a Gateway cloning strategy. Soybean roots were transformed with these constructions using a composite plant system. Confirmation of transformation was attained by PCR and Southern blot analysis. Transgene expression was detected using reverse transcription PCR (RT-PCR) and expression of siRNAs was confirmed in transgenic plants using northern blot analysis. Bioassays performed on transgenic composite plants expressing double-stranded RNA fragments of Cpn-1, Y25 and Prp-17 genes resulted in a 95, 81 and 79% reduction for eggs g(-1) root, respectively. Furthermore, we demonstrated a significant reduction in transcript levels of the Y25 and Prp-17 genes of the nematodes feeding on the transgenic roots via real-time RT-PCR whereas the expression of non-target genes were not affected. The results of this study demonstrate that over-expression of RNA interference constructs of nematode reproduction or fitness-related genes can effectively control H. glycines infection with levels of suppression comparable to conventional resistance.

Rapid in planta evaluation of root expressed transgenes in chimeric soybean plants

Production of stable transgenic plants is one factor that limits rapid evaluation of tissue specific transgene expression. To hasten the assessment of transgenes in planta, we evaluated the use of chimeric soybean seedlings expressing transgenic products in roots. Tap roots from four-day old seedlings (cultivars 'Jack' and KS4704) were excised and hairy roots were induced from hypocotyls via Agrobacterium rhizogenes-mediated transformation. Inoculated hypocotyls were screened on a MS-based medium containing either 200 mg/L kanamycin or 20 mg/L hygromycin. Beta-glucuronidase (GUS) activity assay indicated that highest GUS expression was observed in hypocotyls exposed to a 4-d pre-inoculation time, a neutral pH (7.0) for the co-cultivation medium. A 170-bp of the Fib-1 gene and 292-bp of the Y25C1A.5 gene fragments, both related to nematode reproduction and fitness, were cloned independently into pANDA35HK vector using a Gateway cloning strategy. The resulting RNAi constructs of the genes fragments were transformed into soybean using the chimeric hairy root system and evaluated for its effect on soybean cyst nematode (Heterodera glycines) fecundity. Confirmation of transformation was attained by polymerase chain reaction and Southern-blot analysis, and some potential for suppression of H. glycines reproduction was detected for the two constructs. This method takes on average four weeks to produce chimeric plants ready for transgene analysis.

Effective high-throughput overproduction of membrane proteins in Escherichia coli

Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5 ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-β- d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2 mg/l, with 18 targets producing at levels of 5 mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.

Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets

Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching.

A modified Gateway cloning strategy for overexpressing tagged proteins in plants

BackgroundRecent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale.ResultsHere we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations.ConclusionThis modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins

BackgroundExpression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics.ResultsWith bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies.ConclusionThe DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics.