Cloning Vector Research Articles

Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1)

BackgroundDengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to − 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype.MethodsIn this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1–4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1–4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV).ResultsFrom the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody.ConclusionThis ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1–3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.

Characterization of Cinnamyl Alcohol Dehydrogenase gene family in lignifying tissues of Tectona grandis L.f.

Abstract The cinnamyl alcohol dehydrogenase (CAD) enzyme catalyzes the last step of monolignols synthesis in the lignin pathway. Tectona grandis (teak) is a tropical tree with high valuable tim­ber. As there is few genetic information about lignin formation in teak, the purpose of this study is to characterize members of CAD family in this species. As methodology, PCR amplification using cDNA samples, vector cloning, sequencing, bioinforma­tics analyses and gene expression studies using real time RT-qPCR were performed. As results, four members (TgCAD1- TgCAD4) were obtained. Comparative analyses showed that all of them have conserved residues for catalytic zinc action, structural zinc ligation, NADPH binding and substrate specifici­ty, consistent with the mechanism of alcohol dehydrogenases. Phylogenetic analysis showed that TgCADs are present in three main classes and seven groups. Expression analyses revealed that TgCAD1 was highly expressed in leaves and could be rela­ted with pathogen defense. TgCAD2 was more expressed in branches and roots. Differently, TgCAD3 and TgCAD4 were highly expressed in juvenile and mature sapwood, suggesting a crucial role in wood development and lignin biosynthesis, with tissue-specialized expression profiles. Furthermore, TgCAD4 could be related with teak maturation for being more expressed in sapwood of mature teak trees. As conclusion, this work is the first to characterize genes of CAD family in Tectona grandis. These genes could be interesting to develop transge­nic plants for basic research and field applications.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

Cloning of acetyl-CoA acetyltransferase gene from Halomonas elongata BK-AG18 and in silico analysis of its gene product

Polyhydroxybutyrate (PHB) is a biodegradable polymer that can be used as a substitute for petrochemical plastics. Bacteria accumulate PHB in their cells as carbon and energy reserves because of unbalanced growth conditions. This study aimed to amplify phbA from the chromosomal DNA of Halomonas elongata BK-AG18, a PHB-producing bacterium that was previously isolated from the Bledug Kuwu mud crater of Central Java, Indonesia. The obtained phbA amplicon was 1176 bp. This fragment was cloned into a pGEM-T Easy cloning vector and used to transform Eschericia coli TOP10. The recombinant colonies were selected using blue-white screening, confirmed by size screening, reconfirmed by re-PCR, and sequenced. When putative phbA sequences were aligned with H. elongata DSM2581 chromosome using BLASTN, this sequence showed 99% identity. The deduced amino acid sequences of this clone showed 100% identity to PhbA of H. elongata DSM2581, suggesting that the obtained cloned fragment is a phbA gene. The 3D structure predicted by I-TASSER showed that PhbA of H. elongata BK-AG18 had a high similarity to the acetyl CoA acetyltransferase structure of Ralstonia eutropha H16. PhbA of H. elongata BK-AG18 possesses three catalytic residues, namely Cys88, His348, and Cys378.

Human neonatal Fc receptor as a new potential antibody binding protein for antibody immobilization.

A critical challenge in producing an antibody-based assay with the highest reproducibility and sensitivity is the strategy to immobilize antibodies to solid phase. To date, numerous methods of antibody immobilization were reported but each was subjected to its advantages and limitations. The current study proposes a new potential antibody binding protein, the human neonatal fragment crystallizable (Fc) receptor. This protein has shown its high affinity to the Fc of antibody either in vivo or in vitro. Human neonatal Fc receptor is a heterodimer constructed by p51 α-heavy chain and β2-microglobulin light chain; however, the binding sites toward the antibody are located in the p51 α-heavy chain. Hence, vector cloning and recombinant protein expression were carried out to express the p51 α-heavy chain of the human neonatal Fc receptor (hFcRn-α). The recombinant protein expressed, hFcRn-α, was adopted to pin rabbit IgG against hepatitis B virus surface antigen to a solid phase. A sandwich enzyme-linked immunosorbent assay was further developed to evaluate the efficiency of hFcRn-α-directed immobilization in antigen detection. The result was compared with the conventional physical adsorption method. The findings demonstrated that human neonatal Fc receptor was efficient in pinning antibodies and generating higher signals compared with the physical adsorption of antibody.

First Report of Lily Root Rot Caused by Thanatephorus cucumeris AG 2-1 in the United States

A survey was undertaken on April 2016 to profile soilborne fungal pathogens causing root rot and lesions on lily (Lilium longiflorum) cv. Nellie White in Brookings, Curry County, Oregon. Diseased roots were either blackened or rotted. Sections of diseased roots including the epidermis were surface sterilized and placed on sterile water agar plates containing 100 μg/ml chloramphenicol. From the emerging mycelia, pure cultures were obtained by collecting hyphal tips and placing them on potato dextrose agar (PDA) medium. Recently, we demonstrated the presence of pathogenic Fusarium oxysporum, F. tricinctum, and Ceratobasidium sp. AG-I isolates in eight randomly selected fungal isolates from diseased roots of lilies (Lakshman et al. 2017). In this investigation, three additional fungal isolates showed four to six nuclei per cell under the microscope with lactophenol staining at 400× magnification. The hyphae were branched at right angles, and septa were present near the point of branching. The respective fungal isolates produced dark brown microsclerotia of 2 mm or less in diameter, mostly at the edge of PDA plates. Total DNA from each of the three cultures was PCR amplified with ITS1 and ITS4 ribosomal DNA (rDNA) primers (White et al. 1990) and cloned into a TOPO TA cloning vector (Thermo Fisher Scientific, Waltham, MA). Nucleotide sequences of a randomly chosen clone from each of the three isolates were 706 bp long and identical in sequence. Hence, all were assumed to have originated from a single haplotype and numerated as ELRF 11. The rDNA amplicon deposited in GeneBank (accession no. MF972486) showed 100% homology with the corresponding region of two isolates of Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris) anastomosis group (AG) 2-1 (gb|KP662692 and gb|JQ676881). For pathogenicity tests, 5-mm-diameter plugs from the margin of a 5-day-old culture were placed near the roots of axenically grown lily seedlings, two replicates per treatment and repeated two times, for 10 days at 22°C. Necrotic brown lesions appeared on the roots of such seedlings whereas control inoculation with only PDA plugs did not produce any symptoms on roots. Lactophenol staining and microscopy showed both inner and outer mycelia colonization on infected roots. Additionally, the ELRF 11 caused necrosis and rot upon inoculation of roots of diploid strawberry runners (Hawaii4 F7-3) and necrotic spots on detached leaves of Nicotiana glutinosa, N. benthamiana, and N. paniculata. The onset of necrotic symptoms on those plants occurred 4 to 7 days after inoculation. Fungus with identical morphology was reisolated from root lesions, thus proving Koch’s postulate. No fungus could be isolated from respective roots and leaves of control seedlings. AG 2-1 isolates are reported to be pathogenic on roots, bulbs, and leaves of various hosts, including strawberry and Nicotiana spp. (Yang and Li 2012). Furthermore, both pathogenic and nonpathogenic isolates of AG 2-1 were reported on bulbous crops in the Netherlands (Schneider et al. 1997). To our knowledge, this is the first report of T. cucumeris AG 2-1 causing root rot of lily in the field plots of Oregon or anywhere else in the United States. As various AGs may differ in sensitivity to fungicides and biocontrol agents (Amaradasa et al. 2014), a systematic survey of Rhizoctonia and other pathogens infecting lily under field conditions is required in order to develop effective disease management strategies.

Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly.

Synthetic biology builds upon the foundation of engineering principles, prompting innovation and improvement in biotechnology via a design-build-test-learn cycle. A community-wide standard in DNA assembly would enable bio-molecular engineering at the levels of predictivity and universality in design and construction that are comparable to other engineering fields. Golden Gate Assembly technology, with its robust capability to unidirectionally assemble numerous DNA fragments in a one-tube reaction, has the potential to deliver a universal standard framework for DNA assembly. While current Golden Gate Assembly frameworks (e.g. MoClo and Golden Braid) render either high cloning capacity or vector toolkit simplicity, the technology can be made more versatile—simple, streamlined, and cost/labor-efficient, without compromising capacity. Here we report the development of a new Golden Gate Assembly framework named Mobius Assembly, which combines vector toolkit simplicity with high cloning capacity. It is based on a two-level, hierarchical approach and utilizes a low-frequency cutter to reduce domestication requirements. Mobius Assembly embraces the standard overhang designs designated by MoClo, Golden Braid, and Phytobricks and is largely compatible with already available Golden Gate part libraries. In addition, dropout cassettes encoding chromogenic proteins were implemented for cost-free visible cloning screening that color-code different cloning levels. As proofs of concept, we have successfully assembled up to 16 transcriptional units of various pigmentation genes in both operon and multigene arrangements. Taken together, Mobius Assembly delivers enhanced versatility and efficiency in DNA assembly, facilitating improved standardization and automation.

Cloning of a novel gene from Penicillium oxalicum I1 which in Escherichia coli enhances the secretion of acetic acid

Description of the subject. Organic acids play an important role in the conversion of insoluble ions into soluble ones in soil. Heterologous overexpression of a single gene in a cell is the optimal strategy for increasing the secretion of organic acids solubilizing phosphate. Objectives. In this study, we constructed a primary cDNA library of Penicillium oxalicum I1, and screened clones that can solubilize P in tricalcium phosphate (TCP) medium. We aimed to obtain the gene expressed in Escherichia coli, which can enhance organic acid secretion. Method. A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5'-end of RNA transcription. The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1gene was tested in TCP medium where glucose is the sole carbon source. Afterwards, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium to test the organic acid secretion ability of the transformed E. coli DH5α™. Results. A total of 106 clones showed halos in TCP medium, among which clone I-2 displayed clear halo. The full-length cDNA of clone I-2 was 1,151 bp, with a complete open reading frame of 702 bp, which encoded a hypothetical protein of 233 amino acids. The cDNA sequence showed 68% identity and 73% query cover with other fungal gene sequences of which the function remains unknown. Escherichia coli containing the cloned gene secreted up to 567 mg·l-1 acetic acid within 48 h. The use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid improved the acetic acid secretion of the E. coli DH5α™ clone I-2. By contrast, the use of citric acid, succinic acid, and fumaric acid did not improve the acetic acid secretion of clone I-2 compared to a control E. coli DH5α™ strain bearing only the cloning vector without any insert. Conclusions. We obtained a novel gene from Penicillium oxalicum I1 whose overexpression in E. coli DH5α™ increased the secretion of acetic acid. This observation should help to understand what is the function of the gene isolated from P. oxalicum as well as that of its homologs found in several other species of the Penicillium genus.

A Highly Efficient Strategy for Overexpressing circRNAs.

Circular RNAs (circRNAs) constitute an emerging class of widespread, abundant, and evolutionarily conserved noncoding RNA. They play important and diverse roles in cell development, growth, and tumorigenesis, but functions of the majority of circRNAs remain enigmatic. In order to investigate circRNA function it is necessary to manipulate its expression. While various standard approaches exist for circRNA knockdown, here we present cloning vectors for simplifying the laborious process of cloning circRNAs to achieve high-efficiency overexpression in mammalian cell lines.

Standardized cloning vectors for protein production and generation of large gene libraries in Escherichia coli.

Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.

Cloning and Overexpression Studies on the Glutathione Peroxidase-Like Protein from the Cyanobacterium Nostoc punctiforme ATCC 29133

Cyanobacteria are oxygen-evolving photosynthetic prokaryotes possessing a wide range of enzymatic antioxidants to tackle oxidative stress if and when it arises in cells. Glutathione peroxidase is an important enzyme in the antioxidative machinery of cells and may confer protection to various stress conditions. In the diazotrophic cyanobacterium Nostoc punctiforme ATCC 29133 (hereafter N.punctiforme), an open reading frame (ORF) encoding for a putative glutathione peroxidase was identified as NpunR_4660 by bioinformatics approach. This 486 bp ORF was amplified from the genomic DNA of N.punctiforme by polymerase chain reaction. The forward and reverse primers used for cloning the ORF contained NdeI and XhoI at their 5’ and 3’ end, respectively. The PCR amplified product was sub-cloned into pJET cloning vector. Further the ORF was introduced into pET-20B vector for overexpression of the corresponding protein in Escherichia coli BL21 DE3 strain. After induction of E.coli cultures by Isopropylthiogalactoside (IPTG) and, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of cell-free extracts, an approximately 18 kDa protein was detected in the cytoplasm, which matched with the theoretical molecular weight of the protein. This protein was not present in control cultures transformed with pET-20B without the cyanobacterial ORF. These results suggest that foreign enzymes such as glutathione peroxidase can be produced in large amounts for biotechnological purposes successfully even in cells of a different origin like E.coli.

BiBAC Modification and Stable Transfer into Maize (Zea mays) Hi-II Immature Embryos via Agrobacterium-Mediated Transformation.

Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium-mediated transformation. However, the large size of BiBACs and low-copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning experiments more difficult than other available binary vector systems. Therefore, a protocol that outlines preparation, modification, and transformation of high-molecular weight (HMW) constructs is advantageous for researchers looking to use BiBACs in plant genomics research. This unit does not cover the cloning of HMW DNA into BiBAC vectors. Researchers looking to clone HMW DNA into BiBACs can refer to Zhang etal. (2012; doi: 10.1038/nprot.2011.456). © 2017 by John Wiley & Sons, Inc.

Molecular expression and characterization of GCP7 gene of Haemonchus contortus

Haemonchus contortus is a highly pathogenic and most economically important parasite of sheep and goats worldwide. The cysteine proteases from H. contortus are prime targets for vaccine development. In the present communication we report the molecular expression and characterization of cathepsin B-like cysteine protease, GCP7 gene of H. contortus to study its efficiency as target protein for immunoprophylaxis against haemonchosis in sheep and goats. The complete ORF of GCP7 gene, devoid of the signal sequence, was amplified by RT-PCR from mRNA isolated from H. contortus and was cloned initially into pTZ57R/T cloning vector and then sub-cloned in the pET32a(+) expression vector to produce GCP7 antigen. The nucleotide and deduced amino acid sequence of the GCP7 was aligned against the related sequences of H. contortus available in public domain for in silico analysis by DNA STAR and MEGA version 4.0 softwares. The nucleotide sequence revealed that the GCP7 gene of H.contortus (Indian isolate) encodes 324 amino acids (devoid of signal sequence) and its nucleotide sequence had 95.9% to 99.4% sequence homology with that of U.S.A. and previously published Indian isolates. A high level expression of recombinant (r) GCP7 protein was observed in the molecular range (Mr) of 55 kDa. The rGCP7 protein was confirmed by its specific immunoreactivity against known reference positive sheep sera.

High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing.

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Abstract Background Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica

Effective metabolic engineering of microorganisms relies on balanced expression of both heterologous and endogenous genes to channel metabolic flux towards products of interest while achieving reasonable biomass buildup. To facilitate combinatorial pathway engineering and facile genetic operation, we engineered a set of modular cloning vectors compatible with BioBrick standards, called YaliBricks, to allow for rapid assembly of multigene pathways with customized genetic control elements (promoters, intronic sequences and terminators) in the oleaginous yeast Yarrowia lipolytica. We established a sensitive luciferase reporter and characterized a set of 12 native promoters to expand the oleaginous yeast genetic toolbox for transcriptional fine-tuning. We harnessed the intron alternative splicing mechanism and explored three unique gene configurations that allow us to encode genetic structural variations into metabolic function. We elucidated the role of how these genetic structural variations affect gene expression. To demonstrate the simplicity and effectiveness of streamlined genetic operations, we assembled the 12kb five-gene violacein biosynthetic pathway in one week. We also expanded this set of vectors to accommodate self-cleavage ribozymes and efficiently deliver guide RNA (gRNA) for targeted genome-editing with a codon-optimized CRISPR-Cas9 nuclease. Taken together, the tools built in this study provide a standard procedure to streamline and accelerate metabolic pathway engineering and genetic circuits construction in Yarrowia lipolytica.

CLONING AND EXPRESSION OF Lb-PROTEASE FROM cDNA CLONE OF FOOT-AND- MOUTH DISEASE VIRUS

Foot-and –Mouth disease virus (FMDV) is a positive sense RNA virus and the genome codes for single polyprotein. The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein. The leader L protease which is involved in pathogenesis has two known functions: (i) auto-catalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. To explore the role of L protease in FMDV pathogenesis we generated synthetic FMDV genome lacking the L gene. The gene was amplified from an infectious cDNA clone of serotype Asia1. Primers corresponding to L protease were designed based on the sequence available in the data base. An amplified DNA of 546bp was purified and cloned into pET28 cloning vector. The sequence analysis revealed the presence of single Open Reading Frame (ORF) encoding a protein of 173 amino acid residues. The sequence alignment using BLAST search in NCBI gene Bank showed 91% homology with FMDV strain A isolate IND17/77 L protease gene. The recombinant plasmids pETLb was transferred into BL21 (DE3) pLysS cells and the IPTG induced expressed protein of 25 KDa was purified by nickel affinity column as per the manufacturer’s protocol (Sigma, USA). The specificity of the expressed protein in was confirmed by western blotting using convalescent cattle serum/ rabbit anti-bovine horse radish peroxidase conjugate and O-Dianisidine Dihydrochloride substrate.

Plant Gene Regulation Using Multiplex CRISPR-dCas9 Artificial Transcription Factors.

Besides genome editing, the CRISPR-Cas9-based platform provides a new way of engineering artificial transcription factors (ATFs). Multiplex of guide RNA (gRNA) expression cassettes holds a great promise for many useful applications of CRISPR-Cas9. In this chapter, we provide a detailed protocol for building advanced multiplexed CRISPR-dCas9-Activator/repressor T-DNA vectors for carrying out transcriptional activation or repression experiments in plants. We specifically describe the assembly of multiplex T-DNA vectors that can express multiple gRNAs to activate a silenced gene, or to repress two independent miRNA genes simultaneously in Arabidopsis. We then describe a "higher-order" vector assembly method for increased multiplexing capacity. This higher-order assembly method in principle allows swift stacking of gRNAs cassettes that are only limited by the loading capacity of a cloning or expression vector.

Development of a new host-vector system for colour selection of cloned DNA inserts using a newly designed β-galactosidase gene containing multiple cloning sites in Thermus thermophilus HB27.

We constructed a new Thermus thermophilus cloning vector which enables the colour selection of cloned DNA inserts in the T. thermophilus HB27 host strain (β-gal-) on growth plates containing 3,4-cyclohexenoesculetin β-D-galactopyranoside (S-gal) in the medium. This vector harbors a modified β-galactosidase gene (TTP0042 of T. thermophilus HB27) with 12 unique restriction enzyme sites (Acc65I, AvrII, BlpI, BssHII, EcoRI, EcoRV, HindIII, NruI, SalI, SpeI, SphI and XbaI) as multiple cloning sites under the control of the T. thermophilus slpA promoter. This host-vector system facilitates cloning procedures in T. thermophilus HB27.

Evaluation of pEASY-Uni Seamless Cloning and Assembly Kit to clone multiple fragments of Elaeis guineensis DNA

Several seamless DNA assembly kits based on in vitro homologous recombination activity are commercially available in recent years for efficient and rapid construction of expression vectors, subsequent transformation and investigation of gene functionality. This study was performed to estimate the efficiency of uni seamless cloning system, through cloning of multiple DNA fragments derived from Elaeis guineensis stearoyl-ACP desaturase (SAD) and metallothionein-like protein genes (MET1 and MET2) into a plasmid cloning vector. PCR fragments were assembled based on homologous overlapping regions of 25–30bp into a pUC19 linearized vector. The successful cloning of the three DNA fragments was validated by blue white screening, colony cracking and colony PCR. The observed overall cloning efficiency for the three assembled DNA fragments of about 2.7kb in size was above 65%. This system produces seamless junctions, is directional and does not rely on restriction enzyme digestion and any specific site within the DNA fragments.