The pxst vector was constructed followed the flow chart illustrated in fig. Several versions of broad host range bhr, larabinoseinducible expression vectors were constructed. It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm. Improved gfp and inaz broadhostrange promoterprobe. 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. However, temperature sensitive ts mutations of pbbr1mcs 2 are rarely reported. 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.
The promoterprobe vector petsgfp was made by cloning the bamhikpni fragment containing the green. Brucella abortus strain rb51 as a vector for heterologous. Described here is a set of broad host range plasmids modified for stability in the absence of antibiotic selection and for gene expression manipulation. Improvement of pbbr1mcs plasmids, a very useful series of broad host range cloning vectors. These plasmids are small in size host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes plasmid. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. A plasmid that moves recombinant dna from a test tube back into a cell is an example of a cloning vector, as is a virus that transfers recombinant dna by infection. Tanya soule pk181dmer containing dmer deletion fragment this study pk181dmef containing dmef deletion fragment this study pbbrdmef pbbr1mcs 5 contain entire dmef. 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. Brucella abortus strain rb51 is a stable, rough, attenuated mutant widely used as a live vaccine for bovine brucellosis. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the. As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. Engineering a mevalonate pathway in escherichia coli for production of.
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. Shift in ribonucleotide reductase gene expression in. 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 plasmid vectors for gene expression in.
Based on its complete sequence the plasmid has a size of 41. Cloning vectors cloning vectors are dna molecules that are used to transport cloned sequences between biological hosts and the test tube. Supplementary information an engineered constitutive. First, improved transformation efficiencies of 11 896, 99, 96 and 5989 transformants. A vector is used to amplify a single molecule of dna into many copes. Tisa auniversityofnewhampshire,durham,newhampshire,usa abstract a stable and ef.
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. 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 versatile toolbox for the control of protein levels. We give examples of various wide hostrange plasmid, cosmid, and bac cloning vectors and the types of genes and activities that have been.
Four new derivatives of the broadhostrange cloning. Ampr 6 pbbr1mcs 5 broad host range cloning vector, 4. Pdf broadhostrange plasmid vectors for gene expression. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km gene. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Help pages, faqs, uniprotkb manual, documents, news archive and biocuration projects. It is derived from the broad host range cloning vector pbbr1mcs.
Wide hostrange cloning for functional metagenomics. The broad host range lacz translational fusion vector pmpo200 was constructed to take advantage of the replication and mobilization functions of pbbr1mcs 4 29 and the 5truncated lacz reporter gene from pjes379. 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. This new plasmid has several advantages over pbbr1mcs or its derivatives pbbgroe. 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. Broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 broad host range plasmidmediated metabolic perturbations in pseudomonas fluorescens 525 buch, aditi. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Development of improved versatile broadhostrange vectors.
The xcmiccdbxcmi cassette was amplified by using the pdonr221 vector as a template. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. Kovach me, phillips rw, elzer ph, roop rm, 2nd, peterson km 1994 pbbr1mcs. We cloned four genes for aminoglycoside resistance methyltransferases from the arm and kam families into pbbr1mcs 3 and expressed them in escherichia coli. These plasmids, pbbr1mcs, pbbr1mcs 2, pbbr1mcs 3, pbbr1mcs 4, and pbbr1mcs 5, contain a lacz. Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. Engineering a mevalonate pathway in escherichia coli for. Four new derivatives of the broad host range cloning vector pbbr1mcs. Supplementary table 1 summary of strains and plasmids. 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. Broad host range plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n. 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.
Cyclic digmp modulates the disease progression of erwinia. Broadhostrange plasmidmediated metabolic perturbations. 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. 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. Supplementary table 1 summary of strains and plasmids used in this study strain or plasmid description source li dh10b gibcolife technologies li dym1 ispce. One bacterium for which new genetic tools are needed is the methylotroph methylobacterium extorquens am1. The activity of two of these enzymes was impaired because of the fusion. 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. Construction and characterization of regulated larabinose. Broad host range plasmids fems microbiology letters. Frontiers novel selftransmissible and broadhostrange. These expression vectors were based on a high copy number bhr pbbr1mcs 4 replicon that could replicate in both enteric and nonenteric gramnegative bacteria. 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. Tcr 25 pmb001 to pmb065 sac i fragment containing mtn 5 ss gusa40 insertion from corresponding mb strain cloned into pbbr1mcs 3.
Broad host range expression vectors with tightly regulated. We constructed an improved vector pnsgroe for gene expression studies in brucella spp. Gm r 29 plfc19 edcc gene and native promoter in pbbr1mcs 5. Stable transformation of the actinobacteria frankia spp. Original article open access insights into bacterial co. Broadhostrange expression vectors with tightly regulated. This greatly facilitates genetic studies of a wide variety of gramnegative bacteria and makes pbbr122 particularly interesting for studies of broadhost. Improvement of pbbr1mcs plasmids, a very useful series of. 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. Marx cj, lidstrom me 2002 broad host range crelox system for antibiotic marker recycling in gramnegative bacteria. Broadhostrange plasmids for constitutive and inducible.
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