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Cloning Vectors (cloning + vector)
Selected AbstractsAn in vitro recombination method to convert restriction- and ligation-independent expression vectorsBIOTECHNOLOGY JOURNAL, Issue 3 2008Feng Guo Abstract In recent years, restriction-less recombination cloning systems based on site-specific recombinase with high efficiency have been proven to be very successful. Thus, it is desirable to convert existing conventional vectors to recombination vectors. In this report, we describe the conversion of a set of widely used conventional vectors to Gateway® recombination expression vectors. An attB cassette flanked by several restriction enzyme sites was inserted in a cloning vector, and then subcloned into existing vectors to be converted to construct intermediate vectors containing the attB cassette, which were then converted to recombination expression vectors by in vitro recombination. The intermediate vectors generated in this study can be used for releasing the attB cassette to convert other vectors using the same protocol described here. With the increasing number of recombination vectors constructed with this protocol, the likeliness of releasing the attB cassette from an existing vector, rather than synthesizing it with PCR, will increase. The final expression vectors can also be used for releasing the attR cassette for constructing new vectors. [source] Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone librariesENVIRONMENTAL MICROBIOLOGY, Issue 11 2002Andreas Schramm Summary A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated Td values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. [source] Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and , -galactosidase as selectable markersJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2006V.R. Sridhar Abstract Aims:, We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longum, -galactosidase gene (aglL) as selectable markers. Methods and Results:, The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC,), the resulting vector, pSUW611 (3·9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0·2% loss per generation). The second vector, pSUW711 (5·1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48,72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. Conclusions:, The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. Significance and Impact of the Study:, Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis. [source] Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediationBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009M. Novakova Abstract The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the ,-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants. Biotechnol. Bioeng. 2009;102: 29,37. © 2008 Wiley Periodicals, Inc. [source] Stability of recombinant plasmids on the continuous culture of Bifidobacterium animalis ATCC 27536BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Antonio González Vara Abstract Bifidobacterium animalis ATCC 27536 represents among bifidobacteria a host-model for cloning experiments. The segregational and structural stabilities of a family of cloning vectors with different molecular weights but sharing a common core were studied in continuous fermentation of the hosting B. animalis without selective pressure. The rate of plasmid loss (R) and the specific growth rate difference (,,) between plasmid-free and plasmid-carrying cells were calculated for each plasmid and their relationship with plasmid size was studied. It was observed that both R and the numerical value of ,, increased exponentially with plasmid size. The exponential functions correlating the specific growth rate difference and the rate of plasmid loss with the plasmid molecular weight were determined. Furthermore, the smallest of the plasmids studied, pLAV (4.3-kb) was thoroughly characterized by means of its complete nucleotide sequence. It was found that it contained an extra DNA fragment, the first bifidobacterial insertion sequence characterised, named IS 1999. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 145,150, 2003. [source] | ||||||||||