Pdf broadhostrange plasmid vectors for gene expression in. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. A new and improved hostindependent plasmid system for rk2. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. Four new derivatives of the broadhostrange cloning vector.
An agrobacterium tumefaciens strain with gammaaminobutyric. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibiotic resistance cassettes. However, temperature sensitive ts mutations of pbbr1mcs 2 are rarely reported. 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. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors.
It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm. Pdf broadhostrange plasmid vectors for gene expression. As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. However, temperature sensitive ts mutations of pbbr1mcs2 are rarely reported. The bacterial strains and plasmids used in this study are described in table 1. Upstream of the multiple cloning site of pbbr1mcs 2 is a p lac promoter that typically allows for expression of cloned genes in e. Rhodopseudomonas palustris is a versatile microbe that encounters an innate redox imbalance while growing photoheterotrophically with reduced substrates.
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. Broadhostrange plasmid vectors for gene expression in bacteria. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. 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. Rna sequencing analysis of the broadhostrange strain. Upstream of the multiple cloning site of pbbr1mcs2 is a p lac promoter that typically allows for expression of cloned genes in e. Plasmid pbbr1mcs6y 29 is a gfpexpressing derivative of the broadhostrange cloning vector pbbr1mcs 24. As discussed in materials and methods, the broad hostrange vector pbbr1mcs2 was used as a parental plasmid to construct the phaz overexpression plasmids. Broad host rangeli plasmid vector pbbr1mcs complete. Broadhostrange plasmids for constitutive and inducible. 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.
Brucella abortus strain rb51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. Interactions between brucella melitensis and human phagocytes. Several versions of broad host range bhr, larabinoseinducible expression vectors were constructed. Based on its complete sequence the plasmid has a size of 41. Broadhostrange vector pbbr reso promoter trap vector. New platform for cytochrome p450 reaction combining in.
Kovach m e, elzer p h, hill d s, robertson g t, farris m a, roop r m, peterson k m. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. Belongs to a group of vectors atcc 3762237623, 7728777292 differing only in antibiotic resistance and polylinker orientation. A set of modular broadhostrange expression vectors with various affinity tags sixhistag, flagtag, streptag ii, t7tag was created. 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. Identification and molecular characterization of an efflux. The fosmids used in the conjugation experiments are from a previous study and were constructed using the broadhostrange cloning vector prs44. These plasmids are small in size 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.
Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km gene. Broadhostrange plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 broadhostrange plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 buch, aditi. In contrast to other known broadhostrange vectors, pbbr reso is maintained at a medium copy number and has a reasonable size of about 6. 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. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broadhost. Plasmid pbbr5pemikpkan for constitutive expression c and pbbr5pemikpbad for inducible expression d were derived from gateway cloning compatible broad host range vector pbbr5pemikgw. 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. Thus, we investigated the metabolic perturbations in pseudomonas fluorescens 525 due to the independent. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Broad host range pbbr1based plasmid mutant derivatives.
The resulting excess in reducing equivalents, together with atp from photosynthesis, could be utilized to drive a wide range of bioconversions. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable. National center for biotechnology information general indentifier no. Development and application of a syntheticallyderived. May 16, 2018 a the narrowhostrange vector puc18 possesses several desirable features of a versatile cloning vector. Stable transformation of the actinobacteria frankia spp. The presence of the genes in the cloning vector was confirmed by restriction analysis of the plasmid. Modular broadhostrange expression vectors for single. As opposed to other known broadhostrange vectors, it replicates at medium copy numbers and has a small size 5305 bp. Improvement of pbbr1mcs plasmids, a very useful series of.
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. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer. It was derived from pbbr1mcs, which itself is a modification of the broadhostrange plasmid pbbr1cm. 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 broad host range cloning vectors. Construction and characterization of regulated larabinose. The objective of this study was to genetically modify r. They belong to a family of broad host range cloning and expression vectors pbbr1mcs2 kmr. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broad host. Four new antibioticresistant derivatives of the broadhostrange bhr cloning vector pbbr1mcs have been constructed. Concentration of induced gene product may be regulated by varying the lac inducer concentration. 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.
Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Broadhostrange plasmidmediated metabolic perturbations. Determination of temperature sensitive plasmid copy number in. Sambrook j, russel dw 2001 molecular cloning, a laboratory manual. Broad host range plasmids fems microbiology letters. Frontiers novel selftransmissible and broadhostrange. 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. Thus, we investigated the metabolic perturbations in. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the. 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. 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.
Kovach me, elzer ph, hill ds, robertson gt, farris ma, et al. 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. Determination of temperature sensitive plasmid copy number. 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. Broadhostrange plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. Development and application of a synthetically derived lead. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Four derivatives of the broadhostrange cloning vector pbbr1mcs were successfully introduced into different frankia strains by a filter mating with escherichia coli strain bw29427. The plasmid copy number pcn of temperature sensitive plasmid in host mainly depends on the stability of plasmid.
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. 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. Jul 24, 2007 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes, gene, vol. This plasmid was electroporated into the wildtype 16m and an opsde.
Interactions between brucella melitensis and human. The gentamicin gene was derived from the broadhostrange cloning vector pbbr1mcs5 35. Two identical nonylphenol monooxygenase genes linked to. Mobilization function of the pbhr1 plasmid, a derivative of. Development and application of a syntheticallyderived lead. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Examination of phb depolymerases in ralstonia eutropha. Np5 can degrade a wide range of nonylphenol np isomers that have widely contaminated aquatic environments as major endocrinedisrupting chemicals. Cells were disrupted by sonication, membranes were isolated by ultracentrifugation, analysed by sdspage and western blotting and pa2949 was detected solely in the membrane. Jun 23, 2010 genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids.
Pseudomonas putida u ppu, rfr tipo, cect 4848 was from our collection 7. 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. 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. A the narrowhostrange vector puc18 possesses several desirable features of a versatile cloning vector. 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. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes plasmid.
The biosensors constructed were found to be sensitive, rapid, and speci. Broad host range plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. Mobilization function of the pbhr1 plasmid, a derivative. 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. 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. The broadhostrange plasmid psfa231 isolated from petroleum. These expression vectors were based on a high copy number bhr pbbr1mcs 4 replicon that could replicate in both enteric and nonenteric gramnegative bacteria. Plasmidmediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Brucella abortus strain rb51 as a vector for heterologous.
Recently, it was shown that organic solvents and not antibiotics induce this operon. These new plasmids have several advantages over many of the currently available bhr vectors in that. To construct pyhjh2, the yhjh expression cassette was excised from pyhjh gjermansen et al. Broadhostrange plasmid vectors for gene expression in. Broadhostrange plasmidmediated metabolic perturbations in.
Sensors free fulltext development and application of. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes, gene, vol. For this purpose, the different pcr fragments were ampli. In the present study, the authors characterize a new efflux pump, designated arpabc, on the basis of two isolated chloramphenicolsensitive transposon. The pseudomonas aeruginosa pa14 abc transporter nppa1a2bcd is. Development and application of a synthetically derived. 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.
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