Pbbr1mcs a broad-host-range cloning vector pdf format

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. Despite its importance, few management options are available for diseases caused. This data has been provided by curated databases and other sources that have cited the article. We constructed an improved vector pnsgroe for gene expression studies in brucella spp. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. Cloning and characterization of a tetracycline resistance. However, temperature sensitive ts mutations of pbbr1mcs 2 are rarely reported. As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. Four new cloning vectors have been constructed from the broad host range cloning vector prk290. Host range host range may be wide or narrow broad host range. Improvement of pbbr1mcs plasmids, a very useful series of broad host range cloning vectors. Described here is a set of broad host range plasmids modified for stability in the absence of antibiotic selection and for gene expression manipulation. Pdf detection and validation of a small broadhostrange.

Burkholderia glumae is an emerging plantpathogenic bacterium that causes disease in rice in several of the major riceproducing areas throughout the world. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broadhost. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broad host. These new plasmids have several advantages over many of the currently available bhr vectors in that. They belong to a family of broad host range cloning and expression vectors pbbr1mcs2 kmr. Broad host range plasmids fems microbiology letters oxford. Original article open access insights into bacterial co. Mobilization function of the pbhr1 plasmid, a derivative of the. Construction and characterization of regulated larabinose. Stable transformation of the actinobacteria frankia spp. Broad host range pbbr1based plasmid mutant derivatives. Broadhostrange plasmid vectors for gene expression in. Supplementary information an engineered constitutive. It is capable of transfer, replication, and maintenance in most genera of gramnegative bacteria.

A versatile toolbox for the control of protein levels. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different. 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. 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. 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. Broad host rangeli plasmid vector pbbr1mcs complete. Four new derivatives of the broad host range cloning vector pbbr1mcs. 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. It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm.

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. Different vectors pbbr1cm, pbbr1mcs2, pbbr1mcs4, and pbbr1mcs5 2, 25. 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. 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. Mobilization function of the pbhr1 plasmid, a derivative of. Three broad host range plasmid vectors were selected as starting points for the construction of new plasmidbased expression systems for r. These plasmids are small in size a broad host range promoter cloning vector. 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.

Supplementary table 1 summary of strains and plasmids. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Jul 24, 2007 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes, gene, vol. 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. Marx cj, lidstrom me 2002 broad host range crelox system for antibiotic marker recycling in gramnegative bacteria. Broadhostrange plasmidmediated metabolic perturbations.

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. Nov, 20 broad host vector evaluation and engineering. Factors affecting broad host range bhr plasmid replication. These plasmids, pbbr1mcs, pbbr1mcs 2, pbbr1mcs 3, pbbr1mcs 4, and pbbr1mcs 5, contain a lacz. Twelve clones containing gene fragments induced inside j774 murine. Efficient electrotransformation of corynebacterium diphtheriae with a minireplicon derived from the corynebacterium glutamicum. 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.

Pdf broadhostrange plasmid vectors for gene expression in. Ampr 6 pbbr1mcs 5 broad host range cloning vector, 4. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Broadhostrange plasmid vectors for gene expression in bacteria. 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. Frontiers novel selftransmissible and broadhostrange. Tauch a, kirchner o, loffler b, gotker s, puhler a, kalinowski j 2002. 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. The pxst vector was constructed followed the flow chart illustrated in fig. Brucella abortus strain rb51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. The method, named transmating, should be suited for a broad variety of strains without the need of protocol adaptation for the individual strains. Several versions of broad host range bhr, larabinoseinducible expression vectors were constructed. 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.

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. 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. Improved gfp and inaz broadhostrange promoterprobe. Examples of bacteria in which broadhostrange plasmids with regulatable promoter systems have been used. A schematic overview of the method is given in fig. Concentration of induced gene product may be regulated by varying the lac inducer concentration.

Mobilization function of the pbhr1 plasmid, a derivative. Expression vector construction and introduction in p. Functional metagenomic is a powerful tool that allows the discovery of new enzymes with biotechnological potential. We give examples of various wide hostrange plasmid, cosmid, and bac cloning vectors and the types of genes and activities that have been. Broadhostrange plasmidmediated metabolic perturbations in. The xcmiccdbxcmi cassette was amplified by using the pdonr221 vector as a template.

However, few studies have been conducted to examine the mechanisms underlying the regulation of the lipase expression. These expression vectors were based on a high copy number bhr pbbr1mcs 4 replicon that could replicate in both enteric and nonenteric gramnegative bacteria. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying. For many nonmodel bacterial species, plasmid vectors for gene expression manipulation are unavailable. The use of electrodebased electron acceptors to balance biotransformations may overcome these constraints. The lapg protein plays a role in pseudomonas aeruginosa. Links to pubmed are also available for selected references. Identification of new regulatory genes involved in the. Broadhostrange expression vectors with tightly regulated. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and. 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. Broadhostrange plasmids for constitutive and inducible gene. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km.

The broad host range cloning vector pbbr1mcs 5 was used to create expression vectors of lapd and lapg. 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. Broad host range plasmids fems microbiology letters. A new and improved hostindependent plasmid system for rk2. Four new derivatives of the broadhostrange cloning vector. Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. Development of an inducible lytic system for functional. 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. 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.

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. Broadhostrange plasmids for constitutive and inducible. Bands corresponding to the relaxed form and to the linear form for. 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.

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. Elucidation of oxidation pathways in ralstonia eutropha. Belongs to a group of vectors atcc 3762237623, 7728777292 differing only in antibiotic resistance and polylinker orientation. Constitutive and inducible expression of green fluorescent. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors.

National center for biotechnology information general indentifier no. Broad host range expression vectors with tightly regulated. Tcr 25 pmb001 to pmb065 sac i fragment containing mtn 5 ss gusa40 insertion from corresponding mb strain cloned into pbbr1mcs 3. Supplementary table 1 summary of strains and plasmids used in this study strain or plasmid description source li dh10b gibcolife technologies li dym1 ispce. This new plasmid has several advantages over pbbr1mcs or its derivatives pbbgroe. Primers were designed for unidirectional insertion of the coding sequences between the xbai and hindiii restriction sites of the multiple cloning site. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Cellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 buch, aditi.

Bacteria containing promoter fusions of chromosomal dna to gfp were visualized by fluorescence microscopy and examined by flow cytometry. Loci identification of a nacyl homoserine lactone type. Development of improved versatile broadhostrange vectors. Agrobacterium tumefaciens c58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. Four new derivatives of the broadhostrange cloning vector pbbr1mcs. Bands corresponding to the relaxed form and to the linear form for p2orit. These vectors, pla2901, pla2905, pla2910, and pla2917, confer resistance to kanamycin and tetracycline. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable.

Construction of broadhostrange cosmid cloning vectors. Broadhostrange plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. A bacteriophage vector suitable for sitedirected mutagenesis and highlevel expression of multisubunit proteins in e. 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. Kovach me1, phillips rw, elzer ph, roop rm 2nd, peterson km. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km gene. Enabling unbalanced fermentations by using engineered. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed.

Wide hostrange cloning for functional metagenomics. Improvement of pbbr1mcs plasmids, a very useful series of. Kovach me, elzer ph, hill ds, robertson gt, farris ma, et al. One bacterium for which new genetic tools are needed is the methylotroph methylobacterium extorquens am1. Identification and characterization of three novel esaiesar. The plasmid has been used frequently to design cloning vectors 2, 25. Development of a broadhost synthetic biology toolbox for. 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.

Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. Pseudomonas putida u ppu, rfr tipo, cect 4848 was from our collection 7. The broad host range fosmid and bac vector prs44, fig. Brucella abortus strain rb51 as a vector for heterologous. Four new derivatives of the broadhostrange cloning. Get a printable copy pdf file of the complete article 143k, or click on a page image below to browse page by page. Construction of a broad host range metagenome vector system. Broad host rangeli plasmid vector pmmb67eh complete. Broadhostrange vector pbbr reso promoter trap vector. Full text full text is available as a scanned copy of the original print version. Sambrook j, russel dw 2001 molecular cloning, a laboratory manual. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes plasmid.