As opposed to other known broadhostrange vectors, it replicates at medium copy numbers and has a small size 5305 bp. Mobilization function of the pbhr1 plasmid, a derivative of the. Shift in ribonucleotide reductase gene expression in. 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. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable. A selftransmissible broadhostrange bhr plasmid psfa231 was isolated from petroleumcontaminated sediment in shenfu wastewater irrigation zone, china, using the triparental mating.
The malonate decarboxylase operon of acinetobacter. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. Four new antibioticresistant derivatives of the broadhostrange bhr cloning vector pbbr1mcs have been constructed. 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. Concentration of induced gene product may be regulated by varying the lac inducer. Mobilization function of the pbhr1 plasmid, a derivative. Broad host rangeli plasmid vector pbbr1mcs complete. These new plasmids have several advantages over many of the currently available bhr vectors in that. Plasmid pbbr1mcs6y 29 is a gfpexpressing derivative of the broadhostrange cloning vector pbbr1mcs 24. Gramnegative bacteria and the test of their efficiency using the. The fosmids used in the conjugation experiments are from a previous study and were constructed using the broad host range cloning vector prs44. A new and improved hostindependent plasmid system for rk2.
Genetic determinants involved in the susceptibility of. 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. Following conjugation, the dna fragments of ecoridigested pkp53 from the j532 e. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbrimcs have been con structed. Kovach me1, phillips rw, elzer ph, roop rm 2nd, peterson km. A broadhostrange expression vector series including a. Natural rubber nr or caoutchouc refers to a biopolymer that is synthesized by many plant species and by some fungi. Establishing an innovative carbohydrate metabolic pathway. The resulting plasmid pkp6 was then electroporated into e.
These plasmids typically have a conserved backbone region and a variable accessory region that encodes host beneficial traits. Construction and characterization of regulated larabinose. Diverse broadhostrange plasmids from freshwater carry. For the construction of psrkkm, pbbr1mcs2 was digested with bstbi and. Jul 27, 2019 pbbr1mcs a broadhostrange cloning vector pbbr1mcs a broadhostrange cloning vector pdf whats pbb form a pbb form a pbb 2017 is pbbr2 a solid is pbbr2 a strong electrolyte what is a pbb test. Improved expression vector for brucella species future science. These new plasmids have several advantages over many of the currently available. Transcriptional control of rfah on polysialic and colanic. We used plasmid pbhr1 to test the mobilization host range of pbbr1 derivatives. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors. 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. The three new vectors, psrkkm, psrkgm, and psrktc, all based on the broadhostrange pbbr1mcs vector family, contain a segment that codes for laci q with o 3, the native lac promoter.
These expression vectors were based on a high copy number bhr. Concentration of induced gene product may be regulated by varying the lac inducer concentration. Broad host range plasmids fems microbiology letters. Host range host range may be wide or narrow broad host. Transcriptional control of rfah on polysialic and colanic acid synthesis by escherichia coli k92 nicolas navasa, leandro b. Plasmids containing the same origin of replication pbbr1mcs2 kmr and pbbr1mcs3 tcr have been used to express simultaneous and. This chapter provides methods and insights into the use of broadhostrange plasmid vectors useful for expression of genes in a variety of bacteria. Nr has been commercially exploited for more than 100. Cmr this study pk7 1,156bp nruikpni fragment from pcr product containing promoter, mdcy, and a putative termination dyad symmetric sequence gene and inserted into smaikpni cleavage sites of pbbr1mcs, cmr complementation test. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. 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 manual for the practical study of rootnodule bacteria. Mobilization function of the pbhr1 plasmid, a derivative of.
The engineered pnsgroe expression vector with brucella groe promoter for. Belongs to a group of vectors atcc 3762237623, 7728777292 differing only in antibiotic resistance and polylinker orientation. 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. Metabolic engineering of rhodopseudomonas palustris for. In 1992, antoine and locht reported on pbbr1, a broad host range plasmid.
The promoterprobe vector petsgfp was made by cloning the bamhikpni fragment containing the green. Diverse broadhostrange plasmids from freshwater carry few. Screening of endogenous strong promoters for enhanced. In the case of mucd, transcription was enabled by the lac promoter in pbbr1mcs3. Four new derivatives of the broadhostrange cloning. The fosmids used in the conjugation experiments are from a previous study and. The bacterial strains and plasmids used in this study are described in table 1. Transcriptional control of rfah on polysialic and colanic acid synthesis by escherichia coli k92.
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. Interactions between brucella melitensis and human phagocytes. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm. Cmr this study pk7 1,156bp nruikpni fragment from pcr product containing promoter, mdcy, and a putative termination dyad symmetric sequence gene and inserted into smaikpni cleavage sites of pbbr1mcs, cmr complementation test vector containing mdcy this study. Broad host range plasmids fems microbiology letters oxford. 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.
Frontiers novel selftransmissible and broadhostrange. Previous studies have shown that the broad host range plasmid pbbr1mcs can be used for genetic complementation in brucella abortus. Conjugal vectors for cloning, expression, and insertional. In vivo and in vitro stability of the broadhostrange. Factors affecting broad host range bhr plasmid replication. To extend these observations, the in vivo and in vitro stability of pbbr1mcs was evaluated in the six currently recognized species of the genus brucella. A total of 272 recipients were successfully obtained as plasmid host candidates from granules of an anaerobic methane fermentation plant and from cow manure. The broadhostrange cloning vector pucp27 53 was used to clone the dacb and mpl. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. Host range host range may be wide or narrow broad host range. Engineering a mevalonate pathway in escherichia coli for. 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. Determination of temperature sensitive plasmid copy number. They belong to a family of broad host range cloning and.
Broadhostrange expression vectors with tightly regulated. Sensors free fulltext development and application of. Four new derivatives of the broadhostrange cloning vector. The mostly used promoters in the corresponding expression vector plasmids were the pm promoter, and tac promoter ptac. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm. Novel selftransmissible plasmids were exogenously captured from environmental samples by triparental matings with pbbr1mcs2 as a mobilizable plasmid and pseudomonas resinovorans as a recipient. Pdf detection and validation of a small broadhostrange. One of these metabolic activities, which is h 2 production by. While a wide variety of expression systems are available in e. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. Development of a homologous expression system for rubber. It was derived from pbbr1mcs, which itself is a modification of the broadhostrange plasmid pbbr1cm.
As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. Request pdf improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors pbbr1mcs vectors are small in size. Statistics for comparisons between the groups, independent samples t test, chisquared test. Broad host rangeli plasmid vector pmmb67eh complete. Improvement of pbbr1mcs plasmids, a very useful series of broad. In this study, a small broadhostrange vector named pbbr1mcs2. Statistics for comparisons between the groups, independent samples t test, chisquared test and oneway anova tests were used when appropriate with the spss version 16. When the vector was tested in agrobacterium tumefaciens, a cloned copy of the. The broadhostrange plasmid psfa231 isolated from petroleum. Isolation and molecular characterization of a novel broad. It contains enough genetic information to be mobilized by incp plasmids, to display a medium copy number, and to be stably maintained in all gramnegative bacteria tested to date.
But such vectors remain relatively large, have a low copy number and are therefore unsuitable for some cloning purposes. Growing interest in nonenteric gramnegative bacteria has led to the development of broad host range cloning and expression systems which are mostly based on the replicons of prsf1010 or prk2. Sonja obranic, fedora babic, gordana maravicvlahovicek. Broadhostrange selftransferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. Rhodopseudomonas palustris, which is a member of the purple nonsulfur bacteria group, is often noted for its metabolic versatility.
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 selftransferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. 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. Pdf detection and validation of a small broadhostrange plasmid. Stefan graupner and wilfried wackernagel, a broadhostrange expression vector series including a ptac test plasmid and its application in the expression of the dod gene of serratia marcescens coding for. 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. For example rp4 has unique cloning sites for ecori, bamhi, hindiii, hpai and bglii 5,6 and has been converted into a sali cloning vector prp301 7,8. To assess the strengths of the cloned promoters, the promoters sequences were fused to the 5. Stefan graupner and wilfried wackernagel, a broadhostrange expression vector series including a ptac test plasmid and its application in the expression of the dod gene of serratia marcescens coding for ribulose5phosphate 3epimerase in pseudomonas stutzeri, biomolecular engineering, 17, 1, 11, 2000. The vectors, all based on the broadhostrange plasmid pbbr1, are mobilizable and.
Genetic association of blashv5 with transposable elements. Interactions between brucella melitensis and human. This plasmid was electroporated into the wildtype 16m and an opsde. Improvement of pbbr1mcs plasmids, a very useful series of.
This new plasmid has several advantages over pbbr1mcs. Finally, we have prepared a new expression vector figure 1c. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. For the orthogonality test, the promoters p1 and p2 were assembled with xfp2502 and pta2145 4 combinations on pbbr1mcs2. Broadhostrange plasmid vectors for gene expression in.
Broadhostrange vector pbbr reso promoter trap vector. Based on its complete sequence the plasmid has a size of 41. 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 different antibioticresistance cassettes. The three new vectors, psrkkm, psrkgm, and psrktc, all based on the broadhostrange pbbr1mcs vector family, contain a segment that codes for laci q with o 3, the native lac promoter with o 1, and lacz. 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. Different vectors pbbr1cm, pbbr1mcs2, pbbr1mcs4, and pbbr1mcs5 2, 25 were derived from. In vivo and in vitro stability of the broadhostrange cloning vector pbbr1mcs in six brucella species. Kaori at fumiya, nagmeet na pala noong kabataan pa nila. Jun 22, 2011 this chapter provides methods and insights into the use of broad host range plasmid vectors useful for expression of genes in a variety of bacteria.
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