Cloning Vector Research Articles

Expression of a novel gene encoding protease inhibitor from metagenome of sponge in Vietnam

Marine sponge is known as a “gold mine” of natural products from marine environment. Many novel bioactive compounds have been isolated from marine sponges and sponge-associated microorganisms such as antibiotics, anti-cancer compounds, protease inhibitors, etc. In this study, we selected a gene encoding protease inhibitor from metagenome of a sponge collected in Da Nang to express in Escherichia coli BL21(DE3). The gene PI-DN9 encoding protease inhibitor (1.3 kb) was cut off cloning vector pUC57/PI-DN9 containing gene PI-DN9 and inserted into expression vector pET32a(+), the recombinant vector pET32a(+)/PI-DN9 then was transformed and expressed in the E. coli strain BL21(DE3). Results showed that recombinant protein (50 kDa) was expressed successfully at 25°C, 1 mM of IPTG in 5 hours. The recombinant protein was purified using Ni-NTA affinity chromatography column. Western blot assay and bioactive assay showed good activity of the purified protein.

Effect of Tumor Suppressor MiR-34a Loaded on ZSM-5 Nanozeolite in Hepatocellular Carcinoma: In Vitro and In Vivo Approach

MicroRNA modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC), however Efficient tissue-specific and safe delivery remains a major challenge. We sought to develop an inorganic-organic hybrid vehicle for the systemic delivery of the tumor suppressor miR-34a, and to investigate the efficiency of the delivered miR-34a in the treatment of HCC in vitro and in vivo. In the present study, pEGP-miR cloning and expression vector, expressing miR-34a, was electrostatically bound to polyethyleneimine (PEI), and then loaded onto ZSM-5 zeolite nanoparticles (ZNP). Qualitative and quantitative assessment of the transfection efficiency of miR-34a construct in HepG2 cells was applied by GFP screening and qRT-PCR, respectively. The expression of miR-34a target genes was investigated by qRT-PCR in vitro and in vivo. ZNP/PEI/miR-34a nano-formulation could efficiently deliver into HepG2 cells with low cytotoxicity, indicating good biocompatibility of generated nanozeolite. Furthermore, five injected doses of ZNP/PEI/miR-34a nano-formulation in HCC induced male Balb-c mice, significantly inhibited tumor growth, and demonstrated improved cell structure, in addition to a significant decrease in alphafetoprotein level and liver enzymes activities, as compared to the positive control group. Moreover, injected ZNP/PEI/miR-34a nano-formulation led to a noticeable decrease in the CD44 and c-Myc levels. Results also showed that ZNP/PEI/miR-34a nano-formulation inhibited several target oncogenes including AEG-1, and SOX-9, in vitro and in vivo. Our results suggested that miR-34a is a powerful candidate in HCC treatment and that AEG-1 and SOX-9 are novel oncotargets of miR-34a in HCC. Results also demonstrated that our nano-formulation may serve as a candidate approach for miR-34a restoration for HCC therapy, and generally for safe gene delivery.

Cost-Effective TA Cloning Applied to Sanger Sequencing and HLA Allele Typing

Polymerase chain reaction (PCR)-based technology for clinical HLA typing involves DNA Sanger sequencing of the PCR amplified products of polymorphic loci of HLA, such as HLA-A, -B, -C, -DPB1,-DQB1, and -DRB1, in support of unrelated donor hematopoietic stem cell transplantation. The TA cloning of all the amplicons, followed by Sanger sequencing, provides a primary screening tool of potential organ donors, or to identify individuals who might have potential adverse drug responses. The Class I exon 2 and 3 genes of HLA was amplified as a single fragment and cloned into a TA cloning vector pTZ57R/T, while the Class II exon 2 and 3 amplified fragments were added together in a ligation mix with the vector pTZ57R/T. Positive clones were subjected to Sanger sequencing, and HLA alleles determined using the IMGT database. Results indicate that all the exons of the HLA genes by could be cloned by the strategies described. Furthermore, we were also successful in achieving the amplification of all the desired amplicons as a multiplex PCR, thereby reducing the cost of the modified method further by almost 60 %. We present a cost-effective TA cloning strategy for achieving accurate allele typing of HLA at 2- digit resolution. Taken together, an efficient cloning methodology with a significant lower cost for accurate HLA typing presented here is encouraging. The data suggests that it may be employed for routine cloning of variable targets in molecular biology applications.

Synthesis and molecular cloning of antimicrobial peptide chromogranin A N-46 gene using conventional PCR

BackgroundChromogranin A N-46 (CGA-N46) is an antimicrobial peptide (AMP) consisting of partial N terminus of human chromogranin A. ObjectiveSynthesis and molecular cloning the gene encoding CGA-N46. Materials and methodsThe coding sequence of CGA-N46 gene was synthesized using free bioinformatics tool (Gene2Oligo) that allows the development of in vitro gene synthesis. In this study two methods have been established, stepwise PCR and One Step Simplified Gene Synthesis (SGS) methods. The gene was obtained in stepwise PCR by three steps: 1st PCR to generate short DNA fragments using specific oligonucleotides, 2nd step was the overlap extension PCR (OE-PCR) to bond the short DNA fragments and the final step was the whole gene assembly using amplification primers. Nevertheless, the SGS method was achieved by only one step PCR with various concentrations of oligonucleotides (1000, 500, 250, 100, 50 and 25 nM) and amplification primers (400 nM). In order to achieve a DNA library for future studies, the obtained CGA-N46 gene was cloned into pCR-II TOPO TA cloning vector to create a recombinant vector which was preserved in Escherichia coli (E. coli) TOP10 bacterial stock using transformation process. ResultsThe stepwise PCR method generated a sharp band at expected MW (150 bp). In addition, the SGS method gave one faint band at 1 μM of oligonucleotides. Digestion and PCR analysis revealed that the CGA-N46 gene was successfully cloned in pCR-II TOPO TA cloning vector.

PGP-B2E, a Recombinant Compatible TA/TB-Ligation Vector for Rapid and Inexpensive Gene Cloning.

DNA cloning is the basic step for different fields of life science, and many efforts have been made to simplify this procedure. In this study, we report two general purpose plasmids (pGP), pGP-XB2E and pGP-B2E, for rapid and cost-effective construct of basic clones. The BciVI and XcmI cleavage sites are designed in pGP-XB2E to test plasmid linearization efficiency. The plasmid has better linearization efficiency by using BciVI which could almost completely digest 2μg plasmid in 10min with only one-tenth the recommended enzyme concentration. In order to further optimize the pGP-XB2E, a new plasmid, pGP-B2E, which removed XcmI cleavage site was designed. This vector is highly efficient for cloning PCR products up to 1812bp, and the number of colonies was about five times that of pGP-XB2E. In addition to TA cloning, blunt-end PCR products with T ended in the primer could be positively linked to the T-vector pGP-B2E without A-tailing treatment (TB cloning). Moreover, as an entry vector, pGP-B2E was also compatible for gateway recombination reaction without frameshift mutations. In general, this vector provides a universal, quick, and cost-efficient method for basic molecular cloning.

Abstract GMM-049: STAT3 PROMOTES OVARIAN CANCER GROWTH AND DRUG RESISTANCE BY MODULATING THE ENERGY METABOLISM

Abstract Signal Transducers and Activators of Transcription 3 (STAT3) is a transcription factor that is known to play a key role in cancer progression. In ovarian cancer, STAT3 overexpression leads to increased cancer cell proliferation and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. It is constitutively activated in patient-derived ovarian cancer cells and a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to translocate to mitochondria facilitated by phosphorylation at S727 and modulate mitochondrial function to promote carcinogenesis. Our study aimed to investigate if STAT3 overexpression can modulate cellular metabolism and promote the growth of ovarian cancer cells. We generated stable clones overexpressing STAT3 in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 resulted in increased proliferation, colony formation ability and chemoresistance in vitro and led to large and aggressive ovarian tumors compared to parental and vector controls in xenograft mouse model. STAT3 overexpressing clones exhibited higher mitochondrial respiration and glycolysis placing them in the ‘metabolically active' phenotype compared to parental and vector clones (metabolically less active phenotype). A selective inhibitor of STAT3, Stattic, inhibited both nuclear and mitochondrial STAT3 and also attenuated the STAT3 mediated growth of over-expressing clones both in vitro and in vivo. Stattic treatment also reversed the STAT3-mediated chemoresistance. In contrast, a selective inhibitor of STAT3-Y705, Cryptotanshinone was relatively less effective. Also, Stattic treatments reversed the ‘metabolically active’ state of STAT3 overexpressing clones to a ‘lower metabolic state,’ as the control cells. Stattic also inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200, and OVCAR3) that display a metabolically active phenotype suggesting STAT3 plays a vital role in attaining a metabolically active phenotype by cancer cells. Although, an increase in mitochondrial function was observed in overexpressing A2780 clones as evident from enhanced oxidative phosphorylation, there is no change in the mitochondrial mass or number in overexpressing clones compared to parental A2780 and vector clones indicating the critical role of STAT3 in mitochondrial functions rather than mitochondrial biogenesis in ovarian cancer cells. Further, evaluation of expression and function of mitochondrial STAT3 in ovarian cancers is warranted. Overall, STAT3 can induce metabolic changes in ovarian cancer cells by facilitating mitochondrial function, maybe as a survival mechanism and enhances the cellular fitness of the ovarian cancer cell resulting in increased oncogenic abilities. Citation Format: Vaishnavi Raja, Shailendra Giri, Suhail Hamid, Adnan R Munkarah and Ramandeep Rattan. STAT3 PROMOTES OVARIAN CANCER GROWTH AND DRUG RESISTANCE BY MODULATING THE ENERGY METABOLISM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-049.

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins.

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts.

First Report of Shallot Virus X and Shallot Latent Virus on Shallot (Allium cepa var. aggregatum) in China

Allium species are economically important crops in the world. Viruses are among the most important pathogens affecting their yield (Katis et al. 2012). Only onion yellow dwarf virus (OYDV) has been found in shallot in China (Su et al. 2019). Shallot virus X (SVX, genus Allexivirus, family Alphaflexiviridae) and shallot latent virus (SLV, genus Carlavirus, family Betaflexiviridae) were reported in many Allium species (Chodorska et al. 2014; Kanyuka et al. 1992). In 2018, symptoms consisting of severe leaf twisting, yellowing, and plant stunting were observed on shallot (Allium cepa var. aggregatum) in Jilin Province, China. The symptoms were found in all shallot fields investigated (27), and symptoms were noticed in 20 to 75% of plants. A total of 18 symptomatic plants were collected from nine shallot fields and tested by double antibody sandwich ELISA kit for SVX (DSMZ RT-1042) and TAS-ELISA kit (DSMZ RT-0514-0512/1) for SLV. SVX was detected in two out of 18 shallot samples, and SLV was detected in 15 out of 18 shallot samples. All SVX-positive samples were also SLV positive. The three SVX- and SLV-negative plants were positive for OYDV (using methods as Su et al. 2019) with chlorosis and yellowing. Presence of SVX and SLV in ELISA-positive shallot plants was further confirmed by reverse transcription PCR (RT-PCR). Total RNAs of 18 plant samples were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), reverse transcription and PCR were carried out with the PrimeScript II first Strand cDNA Synthesis Kit (Takara, Japan) and Premix Taq (Ex Taq version 2.0) (Takara). The SVX coat protein (CP) specific primer pair (svxu, 5′-ATGAACGAAGASGATTTGAATCG-3′; svxd, 5′-ARATCGTGKGGATGCATCAGAA-3′) and the SLV CP specific primer pair (slvu, 5′-GGTTCACTTTAGGTTTACAGC-3′; slvd, 5′-GYTTGATCAACATCGATTCTC-3′) designed to amplify complete CP gene of each virus were used for amplification. All SVX ELISA-positive shallot plants yielded an amplicon of the expected size (804 bp), and the SLV-positive plants yielded a 916-bp product each. One RT-PCR product per virus species were sequenced after T vector ligation with the pMD 18-T Vector Cloning Kit (Takara) and submitted to GenBank (accession nos. MK331812 for SVX and MK331810 for SLV). Pairwise comparison of the sequences with other isolates of these viruses available in GenBank showed that CP gene of SVX isolate shared the highest nucleotide/amino acid identities of 86.1/ 94.7 and 86/95.4% with the CP gene of SVX isolates NZ (EU835197) and Russian (JX310755) from New Zealand and Russia, respectively. The CP gene of SLV isolate Nongan-2 shared the highest nucleotide/amino acid identities of 83.0/95.6 and 83.0/95.6% with the CP gene of SLV isolate Xixia (AJ307034) and SLV isolate SLV-WOST87 (LC279526) from China and Japan, respectively. To our knowledge, this is the first report of SVX and SLV infecting shallot in China. All plants with SLV and SVX coinfection showed severe dwarfism symptoms compared with plants with a single infection of SLV. The spread of both viruses could threaten shallot production. Shallot asexual reproduction has been applied for more than 70 years in Jilin and Heilongjiang Provinces; virus detection and virus-free bulb application can avert further spread. Further investigation is necessary to prevent the spread of these pathogens to new locations and to new hosts (Naderi Saffar et al. 2013) in China.

Occurrence and Characterization of Coat Protein Gene of Zucchini yellow mosaic potyvirus (ZYMV) Isolate Infecting Pumpkin (Cucurbita pepo L.) in Bingol Province (Turkey)

Zucchini yellow
 mosaic potyvirus
 (ZYMV) belongs to family Potyviridae, which causes serious economic losses in
 many cucurbits from worldwide. In 2018 (September), leaf samples of pumpkin
 exhibiting typical viral symptoms including mosaic, blistering and wrinkling
 and healthy pumpkin leaf samples were collected from Bingöl and screened by
 Reverse- Transcription Polymerase Chain Reaction (RT-PCR) against ZYMV
 infection. Tested leaf samples were reacted positive resulting in an expected
 about 840 bp DNA fragments of partial coat protein (CP) gene of ZYMV. ZYMV-CP
 gene was further inserted into a pGEM-T Easy prokaryotic cloning vector and
 their partial nucleotide sequences and deduced amino acid were ascertained. The
 provided ZYMV- CP gene sequence consisted of 837 nucleotides in length coded
 for 279 amino acid residues of approximately 31.2 kDa. The isolate was
 denominated as ZYMV-Bingol and registered with MK689858 accession number in the
 NCBI. The sequence of ZYMV- Bingol CP gene were aligned with 21 isolates
 deposited in GenBank from different geographical location and its phylogenetic
 relationships were determined. Molecular analysis of the ZYMV CP gene sequence
 indicated the highest similarity with 100% Turkish isolate (JF317296) and the
 lowest with 91.64% of Korea isolate (AF062518), at nucleotide level. Moreover,
 phylogenetic analyses revealed that ZYMV Bingöl isolate is clustered with the
 Turkish- Adana isolate (JF317296) and Pakistan isolate (AB127936). ZYMV has
 been reported for the first time in the pumpkin plant from Bingöl province of
 Turkey by this study.

Contribution to Carbapenem Resistance and Fitness Cost of DcuS/DcuR, RcsC/RcsB, and YehU/YehT Two-Component Systems in CTX-M-15-Producing Escherichia coli.

Alteration in two-component systems (TCSs), which are signal transduction pathways in prokaryotes, can result in antibiotic resistance. Recently, it has been shown that the overexpression, using a multicopy cloning vector, of the dcuR, rcsB, and yehT genes, which code for the response regulator (RR) part of TCSs, enhanced the minimal inhibitory concentrations (MICs) of carbapenems in Escherichia coli K-12 derivative KAM3. Herein, the contribution to carbapenem resistance of the DcuS/DcuR, RcsC/RcsB, and YehU/YehT TCSs was assessed in E. coli K-12 derivative BW25113 (A phylogroup) and 536 (B2 phylogroup) recipient strains in combination with extended-spectrum β-lactamase that exhibit a weak carbapenemase activity. The genes encoding both the sensor kinase (SK) and the RR, on the one hand, and the genes encoding the SK only, on the other hand, of these regulating pathways were disrupted. Subsequently, the mutants and their parental strains were transformed by a recombinant plasmid encoding the CTX-M-15 gene, before testing their susceptibility to carbapenems and their fitness. Results showed a trade-off between enhanced MICs for ertapenem, which remained above the clinical resistance breakpoint, and decreased growth rate, specifically for the 536 strain SK mutants. In conclusion, mutations in dcuS/dcuR, rcsC/rcsB, and yehU/yehT genes may be a pivotal first-step event in the development of carbapenem resistance.

First Report of Apple Hammerhead Viroid Infecting Apple Trees in South Korea

First Report of Apple Hammerhead Viroid Infecting Apple Trees in South Korea

Cloning and characterization of Rv1980c gene encoding MPT64 Protein from Mycobacterium tuberculosis as a new candidate vaccine of tuberculosis

Pathogenic mycobacteria are one of the major causes of human mortality in the word. Mycobacterium tuberculosis is an etiological agent of human tuberculosis. Designing new vaccines including recombinant protein vaccines may be considered as a new approach for preventing or reducing tuberculosis epidemics. In order to construct protein recombinant as candidate vaccine, the Rv1980c gene encoding MPT64 protein was amplified from M. tuberculosis H37Rv strain genomic DNA using the PCR method and inserted into the cloning vector pGEM-T Easy. The recombinant plasmid pGEM-T Easy-MPT64 was then transformed into E. coli JM109 and cultivated under standard procedure, followed by plasmid extraction, PCR amplification, and DNA sequencing. The correct Rv1980c gene was confirmed by DNA sequencing and subcloned into expression vector pQE30Xa to yield recombinant plasmid pQE30Xa-MPT64, and transformed into E. coli BL21 strain. Transformed white recombinant colony was selected, cultured, induced with 40 μM IPTG, and identified using SDS-PAGE electrophoresis method. The molecular weight was found to be about 24 kDa and identified as recombinant protein MPT64. The target gene has been cloned into host E. coli BL-21 strain and expressed successfully as a soluble protein. The recombinant fusion recombinant protein MPT64 paves the way for tuberculosis diagnosis and vaccine development in the future, especially in Indonesia.

Cloning and expression of recombinant protein CFP21 from Mycobacterium tuberculosis as a tuberculosis vaccine candidate

Increased resistance to TB drugs, may render vaccine development a more effective approach to stop or reduce TB epidemics. The antigen Culture Filtrat Protein Filtrat 21 (CPF21) is an immudominant protein encoded in RD 2 region of the Mycobacterium tuberculosis genome, capable of obtaining a strong hypersensitivity reaction and to induce very high interferon-gamma (IFN-γ) responses in patients with tuberculosis. In order to construct the recombinant plasmid pGEM-T Easy-CFP21 and express it in E. coli BL21, the CFP21 gene was amplified from M. tuberculosis H37Rv genomic DNA using PCR in vitro, and inserted into the pGEM-T Easy cloning vector. The recombinant plasmid was then transformed into E. coli JM109, followed by plasmid extraction, PCR amplification, and DNA sequencing. The correct recombinant CFP21 gene was subcloned into expression vector pGEX-2TK and transformed into E. coli BL21 strain. The white recombinant colony was selected, cultured, induced with 50 µM IPTG, and identified using SDS-PAGE electrophoresis method. These results demonstrated that CFP21 gene has been constructed and expressed successfully. The molecular weight was about 47 kDa as the fusion protein GST-CFP21 and expressed as insoluble protein. In conclusion, the target gene CFP21 has been cloned into host E. coli BL-21 strain and expressed successfully. In the future, the purified recombinant fusion protein GST-CFP21 paves the way for TB diagnosis and vaccine development.

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced \u03b2-galactosidase production using the CRISPR/Cas9 system

The ganA gene from Bacillus subtilis encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.

Attaching Phosphorylated Adaptors/Linkers to Blunt-Ended DNAs.

Adaptors and linkers are attached to the end of DNA fragments to facilitate their insertion into plasmid vectors for cloning and expression.

The Investigation of Drug Resistance Substitutions in NS3 Protease Sequence of Hepatitis C Virus from Non-Responder Patients.

Background:Even with the fantastic successes of direct-acting antivirals (DAA) in the treatment of Hepatitis C Virus (HCV) infection, natural drug resistance remains a challenging obstacle for their impacts. The data regarding protease inhibitors (PIs) resistance in Iran population are limited. The aim of this study was to investigate the variations in NS3 protease of HCV from non-responder patients.Methods:In this cross-sectional study, 14 HCV infected patients with genotype 1(N=5) and 3(N=9) who have not responded to Interferon-related regime were enrolled from Liver Clinic, Shiraz. The NS3 protease region was amplified by Nested-PCR followed by product gel extraction. Besides, some amplified protease regions were cloned into a cloning vector to improve the sensitivity of mutation detection. Both crude and cloned sequences were then introduced into sequencing. The obtained sequences were compared with the NS3 reference sequences and analyzed by Geno2pheno available software to find possible substitutions. In the end, the phylogenetic tree was constructed. Results:Among variations responsible for PIs resistance, only one out of 14 (7%) sample who was infected with genotype 1a, harbored R117C+N174S double mutation, which causes reduced susceptibility to Telaprevir. Any another resistance mutation was not found among the studied population. The most frequent substitutions were determined as I52M(N=9), S102A(N=9), S166A(8) and V170I(8) for genotype 3a, and F147S/A(4) for genotype 1. However, some uncharacterized substitutions on scored position, including I132L(N=1), I170V(N=3) and N174S(N=2) were also determined among sequences. Phylogenetic analysis demonstrated that the protease region has enough power to correctly classify enrolled samples into relevant clusters on the tree. There were 2, 3 and 9 cases of sub-genotypes 1a, 1b, and 3a, respectively.Conclusion:A low frequency of PIs resistance mutations in our HCV infected population is a hopeful point of starting these drugs in HCV infected patients.

English

This study was conducted to determine the heterologous expression of eukaryotic gene in Escherichia coli variable. The expression of Pakistani buffalo (Bubalus bubalis) and cow (Bos taurus) proinsulin genes in BL21 codon plus cells is 30% of total cellular protein. Total RNA was isolated from pancreatic tissues of local (Pakistani) breeds of buffalo (B. bubalis) and cow (B. taurus), converted to cDNA and cloned in a T/A cloning vector. There are five silent mutations: one in B-chain, two each in A-chain and C-peptide encoding regions of local Pakistani buffalo proinsulin DNA sequence as compared to the internationally reported cow proinsulin sequence. The DNA sequence of local (Pakistani) cow proinsulin shows there is one nucleotide encodes C-peptide that has mismatch with the reported cow proinsulin sequence but mismatched nucleotide is same between Pakistani cow and Pakistani buffalo proinsulin sequence. This indicates the genetic similarity of Pakistani cow with Pakistani buffalo. Both genes were expressed in BL21 Codon plus (DE3)-RIL cells with 0.2 mM IPTG at 37°C for 6 to 8 h. Proinsulin was expressed as 30% of total cellular proteins as insoluble inclusion bodies. Key words: Bubalus bubalis, Bos taurus, cDNA, T/A cloning vector, BL21 Codon plus (DE3)-RIL cells.

Novel Expression Vectors Based on the pIGDM1 Plasmid

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIGDM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIGDMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals.

Discovery of the Agrobacterium growth inhibition sequence in virus and its application to recombinant clone screening

Infectious clone vectors used widely in genetic research. While constructing soybean mosaic virus (SMV) clone vectors, we found that transformed Agrobacterium grew significantly different depending on the viral strains used. In particular, the clone vectors constructed with SMV SC15 significantly suppressed the growth of Agrobacterium. Recombinant and truncated virus vector experiments showed that the polymorphism of a P1 protein coding sequence of SC15 leads to the growth inhibition of Agrobacterium. But the lack of other protein encoding sequences, except for the sequence encoding coat protein, should reduce the ability of SC15 to suppress Agrobacterium growth. A vector (pCB301-attL-SC15P) compatible with the Gateway cloning system was constructed using this Agrobacterium inhibitory sequence. The results from the LR recombination reaction with pCB301-attL-SC15P and Agrobacterium transformation showed the valuable application potential of the Agrobacterium inhibitory sequence to serve as a negative screening factor for effective recombinant clone screening in Agrobacterium.

Stable Transformation of the Actinobacteria Frankia spp.

A stable and efficient plasmid transfer system was developed for nitrogen-fixing symbiotic actinobacteria of the genus Frankia, a key first step in developing a genetic system. Four derivatives of the broad-host-range cloning vector pBBR1MCS were successfully introduced into different Frankia strains by a filter mating with Escherichia coli strain BW29427. Initially, plasmid pHKT1 that expresses green fluorescent protein (GFP) was introduced into Frankia casuarinae strain CcI3 at a frequency of 4.0 × 10-3, resulting in transformants that were tetracycline resistant and exhibited GFP fluorescence. The presence of the plasmid was confirmed by molecular approaches, including visualization on agarose gel and PCR. Several other pBBR1MCS plasmids were also introduced into F. casuarinae strain CcI3 and other Frankia strains at frequencies ranging from 10-2 to 10-4, and the presence of the plasmids was confirmed by PCR. The plasmids were stably maintained for over 2 years and through passage in a plant host. As a proof of concept, a salt tolerance candidate gene from the highly salt-tolerant Frankia sp. strain CcI6 was cloned into pBBR1MCS-3. The resulting construct was introduced into the salt-sensitive F. casuarinae strain CcI3. Endpoint reverse transcriptase PCR (RT-PCR) showed that the gene was expressed in F. casuarinae strain CcI3. The expression provided an increased level of salt tolerance for the transformant. These results represent stable plasmid transfer and exogenous gene expression in Frankia spp., overcoming a major hurdle in the field. This step in the development of genetic tools in Frankia spp. will open up new avenues for research on actinorhizal symbiosis.IMPORTANCE The absence of genetic tools for Frankia research has been a major hindrance to the associated field of actinorhizal symbiosis and the use of the nitrogen-fixing actinobacteria. This study reports on the introduction of plasmids into Frankia spp. and their functional expression of green fluorescent protein and a cloned gene. As the first step in developing genetic tools, this technique opens up the field to a wide array of approaches in an organism with great importance to and potential in the environment.