Transcriptional control of rfah on polysialic and colanic. The fosmids used in the conjugation experiments are from a previous study and. Genetic determinants involved in the susceptibility of. Kaori at fumiya, nagmeet na pala noong kabataan pa nila. Improved expression vector for brucella species future science. Plasmid pbbr1 consists of two functional cassettes and presents sequence similarities with the transfer origins of several plasmids and mobilizable. For the construction of psrkkm, pbbr1mcs2 was digested with bstbi and. In 1992, antoine and locht reported on pbbr1, a broad host range plasmid. A new and improved hostindependent plasmid system for rk2.
Pdf detection and validation of a small broadhostrange plasmid. 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. The malonate decarboxylase operon of acinetobacter. 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. Broadhostrange expression vectors with tightly regulated. The promoterprobe vector petsgfp was made by cloning the bamhikpni fragment containing the green.
Belongs to a group of vectors atcc 3762237623, 7728777292 differing only in antibiotic resistance and polylinker orientation. As opposed to other known broad host range vectors, it replicates at medium copy numbers and has a small size 5305 bp. Finally, we have prepared a new expression vector figure 1c. Broadhostrange vector pbbr reso promoter trap vector. 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. The engineered pnsgroe expression vector with brucella groe promoter for. In the case of mucd, transcription was enabled by the lac promoter in pbbr1mcs3. Transcriptional control of rfah on polysialic and colanic acid synthesis by escherichia coli k92 nicolas navasa, leandro b. Host range host range may be wide or narrow broad host range.
Nr has been commercially exploited for more than 100. Plasmids containing the same origin of replication pbbr1mcs2 kmr and pbbr1mcs3 tcr have been used to express simultaneous and. It was derived from pbbr1mcs, which itself is a modification of the broadhostrange plasmid pbbr1cm. Genetic association of blashv5 with transposable elements. A selftransmissible broadhostrange bhr plasmid psfa231 was isolated from petroleumcontaminated sediment in shenfu wastewater irrigation zone, china, using the triparental mating. Shift in ribonucleotide reductase gene expression in. In vivo and in vitro stability of the broadhostrange. 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. While a wide variety of expression systems are available in e. Plasmid pbbr1mcs6y 29 is a gfpexpressing derivative of the broadhostrange cloning vector pbbr1mcs 24.
Interactions between brucella melitensis and human phagocytes. 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. As opposed to other known broadhostrange vectors, it replicates at medium copy numbers and has a small size 5305 bp. In this study, a small broadhostrange vector named pbbr1mcs2. Frontiers novel selftransmissible and broadhostrange. Four new antibioticresistant derivatives of the broadhostrange bhr cloning vector pbbr1mcs have been constructed. We used plasmid pbhr1 to test the mobilization host range of pbbr1 derivatives. 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. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors.
Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. Mobilization function of the pbhr1 plasmid, a derivative. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Gramnegative bacteria and the test of their efficiency using the. Kovach me1, phillips rw, elzer ph, roop rm 2nd, peterson km. Broadhostrange plasmid vectors for gene expression in.
Transcriptional control of rfah on polysialic and colanic acid synthesis by escherichia coli k92. A broadhostrange expression vector series including a. Determination of temperature sensitive plasmid copy number. This new plasmid has several advantages over pbbr1mcs. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host beneficial traits. This plasmid was electroporated into the wildtype 16m and an opsde.
The bacterial strains and plasmids used in this study are described in table 1. 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. Interactions between brucella melitensis and human. 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. 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. Rhodopseudomonas palustris, which is a member of the purple nonsulfur bacteria group, is often noted for its metabolic versatility. Broad host rangeli plasmid vector pmmb67eh complete. Screening of endogenous strong promoters for enhanced. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Concentration of induced gene product may be regulated by varying the lac inducer concentration. 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. Pdf detection and validation of a small broadhostrange. 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. To assess the strengths of the cloned promoters, the promoters sequences were fused to the 5.
Previous studies have shown that the broad host range plasmid pbbr1mcs can be used for genetic complementation in brucella abortus. Concentration of induced gene product may be regulated by varying the lac inducer. Mobilization function of the pbhr1 plasmid, a derivative of the. It was derived from pbbr1mcs, which itself is a modification of the broad host range plasmid pbbr1cm. Following conjugation, the dna fragments of ecoridigested pkp53 from the j532 e. Improvement of pbbr1mcs plasmids, a very useful series of broad.
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. 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. 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. These expression vectors were based on a high copy number bhr pbbr1mcs 4 replicon that could replicate in both enteric and nonenteric gramnegative bacteria. Diverse broadhostrange plasmids from freshwater carry. 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. Novel selftransmissible plasmids were exogenously captured from environmental samples by triparental matings with pbbr1mcs2 as a mobilizable plasmid and pseudomonas resinovorans as a recipient.
They belong to a family of broad host range cloning and. For the orthogonality test, the promoters p1 and p2 were assembled with xfp2502 and pta2145 4 combinations on pbbr1mcs2. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. Broadhostrange selftransferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts.
Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Mobilization function of the pbhr1 plasmid, a derivative of. 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. Metabolic engineering of rhodopseudomonas palustris for. Broad host range plasmids fems microbiology letters. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. The broadhostrange plasmid psfa231 isolated from petroleum. Improvement of pbbr1mcs plasmids, a very useful series of. Several versions of broad host range bhr, larabinoseinducible expression vectors were constructed. These new plasmids have several advantages over many of the currently available bhr vectors in that. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km.
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. Broad host range selftransferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. 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. Host range host range may be wide or narrow broad host. Natural rubber nr or caoutchouc refers to a biopolymer that is synthesized by many plant species and by some fungi. 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. Request pdf improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors pbbr1mcs vectors are small in size.
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. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. These expression vectors were based on a high copy number bhr. Establishing an innovative carbohydrate metabolic pathway. Four new derivatives of the broadhostrange cloning vector. Factors affecting broad host range bhr plasmid replication. When the vector was tested in agrobacterium tumefaciens, a cloned copy of the. The mostly used promoters in the corresponding expression vector plasmids were the pm promoter, and tac promoter ptac. Different vectors pbbr1cm, pbbr1mcs2, pbbr1mcs4, and pbbr1mcs5 2, 25 were derived from.
Isolation and molecular characterization of a novel broad. 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. But such vectors remain relatively large, have a low copy number and are therefore unsuitable for some cloning purposes. Construction and characterization of regulated larabinose. Broad host rangeli plasmid vector pbbr1mcs complete. Broad host range plasmids fems microbiology letters oxford.
Engineering a mevalonate pathway in escherichia coli for. 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. 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. The resulting plasmid pkp6 was then electroporated into e. Statistics for comparisons between the groups, independent samples t test, chisquared test. Four new derivatives of the broadhostrange cloning. Sensors free fulltext development and application of. These new plasmids have several advantages over many of the currently available. In vivo and in vitro stability of the broadhostrange cloning vector pbbr1mcs in six brucella species. This chapter provides methods and insights into the use of broadhostrange plasmid vectors useful for expression of genes in a variety of bacteria. Development of a homologous expression system for rubber. Conjugal vectors for cloning, expression, and insertional.
The vectors, all based on the broadhostrange plasmid pbbr1, are mobilizable and. 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. Diverse broadhostrange plasmids from freshwater carry few. 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. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm. 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. One of these metabolic activities, which is h 2 production by. Based on its complete sequence the plasmid has a size of 41. A manual for the practical study of rootnodule bacteria. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbrimcs have been con structed.
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