A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. However, temperature sensitive ts mutations of pbbr1mcs2 are rarely reported. Development and application of a syntheticallyderived lead. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes plasmid. Broadhostrange plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. Plasmid pbbr5pemikpkan for constitutive expression c and pbbr5pemikpbad for inducible expression d were derived from gateway cloning compatible broad host range vector pbbr5pemikgw. The gentamicin gene was derived from the broadhostrange cloning vector pbbr1mcs5 35.
Broad host range plasmids fems microbiology letters. Brucella abortus strain rb51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Upstream of the multiple cloning site of pbbr1mcs2 is a p lac promoter that typically allows for expression of cloned genes in e. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes.
Interactions between brucella melitensis and human phagocytes. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. Determination of temperature sensitive plasmid copy number in. 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. This is the first report on dual synthesis of polyr3hydroxybutyric acid phb and protein polymer by recombinant microorganisms for use in. New platform for cytochrome p450 reaction combining in. These expression vectors were based on a high copy number bhr pbbr1mcs 4 replicon that could replicate in both enteric and nonenteric gramnegative bacteria. Determination of temperature sensitive plasmid copy number. Frontiers novel selftransmissible and broadhostrange. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broadhost. Kovach me, elzer ph, hill ds, robertson gt, farris ma, et al. In this study, a small broadhostrange vector named pbbr1mcs2, 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 broadhostrange vectors, pbbr reso is maintained at a medium copy number and has a reasonable size of about 6.
Pdf broadhostrange plasmid vectors for gene expression. 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. May 16, 2018 a the narrowhostrange vector puc18 possesses several desirable features of a versatile cloning vector. The broadhostrange plasmid psfa231 isolated from petroleum. Broadhostrange plasmid vectors for gene expression in. Upstream of the multiple cloning site of pbbr1mcs 2 is a p lac promoter that typically allows for expression of cloned genes in e. The gabt fragment was subcloned into the multiple cloning site of the broadhostrange plasmid pbbr1mcs5 38 using hindiii and xbai new england biolabs, hirchin, uk to generate a laczgabt. Two identical nonylphenol monooxygenase genes linked to. To understand the biochemical and genetic backgrounds of np degradation, a gene library of strain np5 was constructed using a broadhostrange vector pbbr1mcs2 and introduced into sphingobium. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors. Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Broad host range plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. National center for biotechnology information general indentifier no. Concentration of induced gene product may be regulated by varying the lac inducer concentration.
Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. An agrobacterium tumefaciens strain with gammaaminobutyric. Broadhostrange vector pbbr reso promoter trap vector. 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.
Broadhostrange plasmid vectors for gene expression in bacteria. Kovach m e, elzer p h, hill d s, robertson g t, farris m a, roop r m, peterson k m. Four new derivatives of the broadhostrange cloning vector. Development and application of a synthetically derived lead. Plasmidmediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Improvement of pbbr1mcs plasmids, a very useful series of broad host range cloning vectors. Novel selftransmissible plasmids were exogenously captured from environmental samples by triparental matings with pbbr1mcs2 as a mobilizable plasmid and pseudomonas resinovorans as a recipient.
Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibiotic resistance cassettes. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Broad host range plasmid pbbr1mcs2 derived from pbbr1 is mobilizable and compatible with various plasmid incompatibility groups and wildly used for cloning and expression in laboratory. 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. A selftransmissible broad host range bhr plasmid psfa231 was isolated from petroleumcontaminated sediment in shenfu wastewater irrigation zone, china, using the triparental mating exogenous plasmid capture method. The bacterial strains and plasmids used in this study are described in table 1.
The objective of this study was to genetically modify r. However, temperature sensitive ts mutations of pbbr1mcs 2 are rarely reported. Broadhostrange plasmidmediated metabolic perturbations in. Construction and characterization of regulated larabinose. Mobilization function of the pbhr1 plasmid, a derivative of. The gabt fragment was subcloned into the multiple cloning site of the broad host range plasmid pbbr1mcs 5 38 using hindiii and xbai new england biolabs, hirchin, uk to generate a laczgabt. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. Brucella abortus strain rb51 as a vector for heterologous. A set of modular broadhostrange expression vectors with various affinity tags sixhistag, flagtag, streptag ii, t7tag was created. For this purpose, the different pcr fragments were ampli. Thus, we investigated the metabolic perturbations in pseudomonas fluorescens 525 due to the independent. These new plasmids have several advantages over many of the currently available bhr vectors in that.
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. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. In the present study, the authors characterize a new efflux pump, designated arpabc, on the basis of two isolated chloramphenicolsensitive transposon. Stable transformation of the actinobacteria frankia spp. Read novel broad host range shuttle vectors for expression in escherichia coli, bacillus subtilis and pseudomonas putida, journal of biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at. The authors previously described srpabc, an operon involved in protondependent solvent efflux in the solventtolerant pseudomonas putida s12.
The fosmids used in the conjugation experiments are from a previous study and were constructed using the broadhostrange cloning vector prs44. It was derived from pbbr1mcs, which itself is a modification of the broadhostrange plasmid pbbr1cm. 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. Identification and molecular characterization of an efflux. Broadhostrange plasmidmediated metabolic perturbations. Recently, it was shown that organic solvents and not antibiotics induce this operon. 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. As discussed in materials and methods, the broad host range vector pbbr1mcs 2 was used as a parental plasmid to construct the phaz overexpression plasmids. As discussed in materials and methods, the broad hostrange vector pbbr1mcs2 was used as a parental plasmid to construct the phaz overexpression plasmids. In order to overcome this problem, we introduced by sitedirected mutagenesis a new ndei restriction site into pbbr1mcs3 to generate a start codon directly at the beginning. 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.
Broadhostrange plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 broadhostrange plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 buch, aditi. Four new derivatives of the broadhostrange cloning. Pseudomonas putida u ppu, rfr tipo, cect 4848 was from our collection 7. The pseudomonas aeruginosa pa14 abc transporter nppa1a2bcd is. Development and application of a syntheticallyderived. Jul 24, 2007 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes, gene, vol. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer. As opposed to other known broadhostrange vectors, it replicates at medium copy numbers and has a small size 5305 bp. Pdf broadhostrange plasmid vectors for gene expression in. Plasmid pbbr4pemik for restriction cloning a and pbbr4pemikgw for gateway cloning invitrogen b were derived from broad host range vector pbbr1mcs4, which carries ampicillin resistance. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km gene. 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. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broad host. 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.
Rhodopseudomonas palustris is a versatile microbe that encounters an innate redox imbalance while growing photoheterotrophically with reduced substrates. To construct pyhjh2, the yhjh expression cassette was excised from pyhjh gjermansen et al. Np5 can degrade a wide range of nonylphenol np isomers that have widely contaminated aquatic environments as major endocrinedisrupting chemicals. This plasmid was electroporated into the wildtype 16m and an opsde. Interactions between brucella melitensis and human. Four derivatives of the broadhostrange cloning vector pbbr1mcs were successfully introduced into different frankia strains by a filter mating with escherichia coli strain bw29427. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. They belong to a family of broad host range cloning and expression vectors pbbr1mcs2 kmr. Several versions of broad host range bhr, larabinoseinducible expression vectors were constructed. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes, gene, vol.
The presence of the genes in the cloning vector was confirmed by restriction analysis of the plasmid. Broad host rangeli plasmid vector pmmb67eh complete. Oct 15, 2012 read novel broad host range shuttle vectors for expression in escherichia coli, bacillus subtilis and pseudomonas putida, journal of biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Development and application of a synthetically derived. The resulting excess in reducing equivalents, together with atp from photosynthesis, could be utilized to drive a wide range of bioconversions. Modular broadhostrange expression vectors for single. Examination of phb depolymerases in ralstonia eutropha. Thus, we investigated the metabolic perturbations in. The biosensors constructed were found to be sensitive, rapid, and speci. Based on its complete sequence the plasmid has a size of 41. Mobilization function of the pbhr1 plasmid, a derivative. Plasmid pbbr1mcs6y 29 is a gfpexpressing derivative of the broadhostrange cloning vector pbbr1mcs 24. Temperature sensitive plasmid pbbr1mcs2ts, mutated from pbbr1mcs and derived from pbbr1, is a broad host range plasmid and it is especially useful for gene targeting and integration in various hosts.
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 plasmid copy number pcn of temperature sensitive plasmid in host mainly depends on the stability of plasmid. A the narrowhostrange vector puc18 possesses several desirable features of a versatile cloning vector. Rna sequencing analysis of the broadhostrange strain. It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm. A selftransmissible broadhostrange bhr plasmid psfa231 was isolated from petroleumcontaminated sediment in shenfu wastewater irrigation zone, china, using the triparental mating exogenous plasmid capture method. Metabolic engineering of rhodopseudomonas palustris for. Improvement of pbbr1mcs plasmids, a very useful series of. Sensors free fulltext development and application of. A new and improved hostindependent plasmid system for rk2. Broad host rangeli plasmid vector pbbr1mcs complete. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Broadhostrange plasmids for constitutive and inducible. Belongs to a group of vectors atcc 3762237623, 7728777292 differing only in antibiotic resistance and polylinker orientation.
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