Pbbr1mcs a broad-host-range cloning vector pdf format

Get a printable copy pdf file of the complete article 143k, or click on a page image below to browse page by page. This data has been provided by curated databases and other sources that have cited the article. A schematic overview of the method is given in fig. Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. A gene fusion system based on plasmid pbbr1mcs and the expression of green fluorescent protein was developed for brucella suis, allowing isolation of constitutive and inducible genes. Cloning and characterization of a tetracycline resistance. Improvement of pbbr1mcs plasmids, a very useful series of. Kovach me1, phillips rw, elzer ph, roop rm 2nd, peterson km. We constructed an improved vector pnsgroe for gene expression studies in brucella spp. Loci identification of a nacyl homoserine lactone type. Jul 24, 2007 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes, gene, vol.

Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Tauch a, kirchner o, loffler b, gotker s, puhler a, kalinowski j 2002. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors. Broad host range pbbr1based plasmid mutant derivatives. Supplementary table 1 summary of strains and plasmids used in this study strain or plasmid description source li dh10b gibcolife technologies li dym1 ispce. Ampr 6 pbbr1mcs 5 broad host range cloning vector, 4. Broadhostrange plasmids for constitutive and inducible gene. Four new derivatives of the broadhostrange cloning.

Pseudomonas putida u ppu, rfr tipo, cect 4848 was from our collection 7. Constitutive and inducible expression of green fluorescent. In order to overcome this problem, we introduced by sitedirected mutagenesis a new ndei restriction site into pbbr1mcs 3 to generate a start codon directly at the beginning. Agrobacterium tumefaciens c58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. Using broad host range replicons as the basis of cloning, vector development has the advantage that the cloning may be performed by standard techniques in e. Nov, 20 broad host vector evaluation and engineering. Bands corresponding to the relaxed form and to the linear form for p2orit. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. These plasmids, pbbr1mcs, pbbr1mcs 2, pbbr1mcs 3, pbbr1mcs 4, and pbbr1mcs 5, contain a lacz.

The plasmid has been used frequently to design cloning vectors 2, 25. The pxst vector was constructed followed the flow chart illustrated in fig. Mobilization function of the pbhr1 plasmid, a derivative of. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. For many nonmodel bacterial species, plasmid vectors for gene expression manipulation are unavailable. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Novel selftransmissible plasmids were exogenously captured from environmental samples by triparental matings with pbbr1mcs 2 as a mobilizable plasmid and pseudomonas resinovorans as a recipient. Bacteria containing promoter fusions of chromosomal dna to gfp were visualized by fluorescence microscopy and examined by flow cytometry. We give examples of various wide hostrange plasmid, cosmid, and bac cloning vectors and the types of genes and activities that have been. Pdf detection and validation of a small broadhostrange. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. The use of electrodebased electron acceptors to balance biotransformations may overcome these constraints.

It is capable of transfer, replication, and maintenance in most genera of gramnegative bacteria. Links to pubmed are also available for selected references. Four new derivatives of the broadhostrange cloning vector. Broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 buch, aditi. Examples of bacteria in which broadhostrange plasmids with regulatable promoter systems have been used. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable. The vectors, all based on the broadhostrange plasmid pbbr1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha and gammaproteobacteria. However, few studies have been conducted to examine the mechanisms underlying the regulation of the lipase expression. However, temperature sensitive ts mutations of pbbr1mcs 2 are rarely reported. The xcmiccdbxcmi cassette was amplified by using the pdonr221 vector as a template. 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.

Broad host rangeli plasmid vector pmmb67eh complete. Incq and small incp vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small incp plasmid was. Plasmids with rk2 ori region will replicate in most gram negative bacteria plasmids with the rsf 1010 ori can replicate in gram negative and gram. One bacterium for which new genetic tools are needed is the methylotroph methylobacterium extorquens am1. Broadhostrange plasmid vectors for gene expression in bacteria. Despite its importance, few management options are available for diseases caused. Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. Jun 23, 2010 read broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525, applied microbiology and biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Original article open access insights into bacterial co. Concentration of induced gene product may be regulated by varying the lac inducer concentration. Improved gfp and inaz broadhostrange promoterprobe. Broadhostrange plasmidmediated metabolic perturbations in. The method, named transmating, should be suited for a broad variety of strains without the need of protocol adaptation for the individual strains.

Broadhostrange plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. Several versions of broad host range bhr, larabinoseinducible expression vectors were constructed. Pdf broadhostrange plasmid vectors for gene expression in. Tcr 25 pmb001 to pmb065 sac i fragment containing mtn 5 ss gusa40 insertion from corresponding mb strain cloned into pbbr1mcs 3. Brucella abortus strain rb51 as a vector for heterologous. Identification of new regulatory genes involved in the. The lapg protein plays a role in pseudomonas aeruginosa. Due to the fact that many gramnegative bacteria are naturally cm r, four derivatives of the vector were constructed, each with a different antibiotic selection marker. Four new cloning vectors have been constructed from the broad host range cloning vector prk290.

Primers were designed for unidirectional insertion of the coding sequences between the xbai and hindiii restriction sites of the multiple cloning site. Development of an inducible lytic system for functional. Recently, a set of broad host range cloning vectors derived from the bordetella bronchiseptica plasmid pbbr1 antoine and locht 1992 was described kovach et al. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and. Plasmid vectors are a valuable research tool for characterizing bacterial gene function, but there is a limited range of plasmids that are functional in nonmodel bacterial species. A stable and efficient plasmid transfer system was developed for nitrogenfixing symbiotic actinobacteria of the genus frankia, a key first step in developing a genetic system. Broadhostrange plasmids for constitutive and inducible. Burkholderia glumae is an emerging plantpathogenic bacterium that causes disease in rice in several of the major riceproducing areas throughout the world. Described here is a set of broad host range plasmids modified for stability in the absence of antibiotic selection and for gene expression manipulation. Broadhostrange plasmidmediated metabolic perturbations. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying.

Broad host range expression vectors with tightly regulated. Factors affecting broad host range bhr plasmid replication. They belong to a family of broad host range cloning and expression vectors pbbr1mcs2 kmr. Development of improved versatile broadhostrange vectors. The three new vectors, psrkkm, psrkgm, and psrktc, all based on the broad host range pbbr1mcs vector family, contain a segment that codes for laci q with o 3, the native lac promoter with o 1, and lacz. Mobilization function of the pbhr1 plasmid, a derivative of the. Efficient electrotransformation of corynebacterium diphtheriae with a minireplicon derived from the corynebacterium glutamicum. Supplementary table 1 summary of strains and plasmids.

Supplementary information an engineered constitutive. The rk2 plasmid is a broad host range plasmid belonging to the incp incompatibility group it is notable for its ability to replicate in a wide variety of singlecelled organisms, which makes it suitable as a genetic engineering tool. A versatile toolbox for the control of protein levels. Enabling unbalanced fermentations by using engineered. These new plasmids have several advantages over many of the currently available bhr vectors in that. In this study, a small broad host range vector named pbbr1mcs 2, which does not belong to the incq, incw, or incp groups, was studied for the feasibility of its use in conjugative gene transfer. In contrast to other known broad host range vectors, pbbr reso is maintained at a medium copy number and has a reasonable size of about 6. We describe how wide hostrange cloning vectors can lead to more flexible and effective procedures to isolate novel genes by screening metagenomic libraries in a range of bacterial hosts, not just the conventionally used escherichia coli. Construction and characterization of regulated larabinose. Broad host rangeli plasmid vector pbbr1mcs complete.

Elucidation of oxidation pathways in ralstonia eutropha. The seva platform involves the adoption of a plain set of rules for the physical assembly of the three basic components of plasmid vectors i. Marx cj, lidstrom me 2002 broad host range crelox system for antibiotic marker recycling in gramnegative bacteria. A bacteriophage vector suitable for sitedirected mutagenesis and highlevel expression of multisubunit proteins in e. Gmr 6 pbbr1mcs 2 ackrst broad host range cloning vector, contains the acetyl lysyltrna synthetase pyls under control of p glns and the suppressor trna cua pylt under the control of p prok, inserted into the nsii.

These expression vectors were based on a high copy number bhr pbbr1mcs 4 replicon that could replicate in both enteric and nonenteric gramnegative bacteria. Improvement of pbbr1mcs plasmids, a very useful series of broad host range cloning vectors. We observed that a mutation affecting a trna processing function significantly affected the emergence of such mutants, suggesting that c58 contained a positively acting gene conferring resistance to tetracycline. Kovach me, elzer ph, hill ds, robertson gt, farris ma, et al. A new and improved hostindependent plasmid system for rk2. Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Broad host range plasmids fems microbiology letters oxford. Mobilization function of the pbhr1 plasmid, a derivative. Broadhostrange plasmid vectors for gene expression in. Different vectors pbbr1cm, pbbr1mcs2, pbbr1mcs4, and pbbr1mcs5 2, 25. These vectors, pla2901, pla2905, pla2910, and pla2917, confer resistance to kanamycin and tetracycline.

Three broad host range plasmid vectors were selected as starting points for the construction of new plasmidbased expression systems for r. Broad host range plasmids fems microbiology letters. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. Construction of a broad host range metagenome vector system. Four new derivatives of the broad host range cloning vector pbbr1mcs. Full text full text is available as a scanned copy of the original print version. Our ultimate goal is to develop strain rb51 as a preferential vector for the delivery of protective antigens of other intracellular pathogens to which the induction of a strong th1 type of immune response is needed for effective protection. A multipurpose broad host range plasmid, pij3200, was constructed by inserting the polylinkercontaining 445 by pvuii fragment of bluescript m into the ecori site of the cosmid plafr1. Host range host range may be wide or narrow broad host range plasmids will often encode all genes necessary for initiating replication promoters and proteins must be adapted to work in host bacteria mostly determined by origin of replication e. As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. The broad host range fosmid and bac vector prs44, fig. This new plasmid has several advantages over pbbr1mcs or its derivatives pbbgroe.

Expression vector construction and introduction in p. Bands corresponding to the relaxed form and to the linear form for. The broad host range cloning vector pbbr1mcs 5 was used to create expression vectors of lapd and lapg. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the. It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different. National center for biotechnology information general indentifier no. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broad host. Broad host range plasmid pbbr1mcs 2 derived from pbbr1 is mobilizable and compatible with various plasmid incompatibility groups and wildly used for cloning and expression in laboratory. Broadhostrange vector pbbr reso promoter trap vector. Stable transformation of the actinobacteria frankia spp. Sambrook j, russel dw 2001 molecular cloning, a laboratory manual.

Broadhostrange expression vectors with tightly regulated. Each vector contains a versatile multiple cloning site that includes an ndei site allowing fusion of the cloned gene to the initiation codon of lacz. Brucella abortus strain rb51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Construction of broadhostrange cosmid cloning vectors. Twelve clones containing gene fragments induced inside j774 murine. Identification and characterization of three novel esaiesar. It is derived from the broad host range cloning vector pbbr1mcs. Wide hostrange cloning for functional metagenomics. These plasmids are small in size a broad host range promoter cloning vector. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. Frontiers novel selftransmissible and broadhostrange. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km gene. Development of a broadhost synthetic biology toolbox for. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broadhost.