Broadhostrange vector pbhr1 mobilizable pbhr1 is a mobilizable vector that was created by removing a frame shift in the mobilization gene of pbbr122, thereby. It is derived from rsf1010, a naturally occurring broad host range vector found in e. Host range host range may be wide or narrow broad host range. The wild type plasmid carrying the curing cassette displaces target plasmids poorly. Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes.
Concentration of induced gene product may be regulated by varying the lac inducer. Four new derivatives of the broadhostrange cloning. But such vectors remain relatively large, have a low copy number and are therefore unsuitable for some cloning purposes. The 393base pair minimal origin,oriv, of plasmid rk2 contains three iterated motifs essential for initiation of replication. Convenient broadhostrange unstable vectors for studying. Next generation soil metagenomics largeinsert bac libraries for the discovery of natural products and drugs abstract mark liles2, david mead 1, xing cong li3, kavita kakirde2, rosa ye1, megan wagner, amanda krerowicz, molly staley2, svetlana jasinovica1, melissa r jacob3, ameeta kagarwal3, peter ladell1. These new plasmids have several advantages over many of the currently available bhr vectors in that. Improvement of pbbr1mcs plasmids, a very useful series of. Next generation soil metagenomics largeinsert bac libraries. Here, we introduce a series of new rk2 based broad host range expression plasmids for inducible production of affinity and fluorescence tagged proteins in the cytoplasm and. The multi host vector prl83a was used in this study. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used. It is possible that this organization contributes to an initiation mechanism which allows. The main aim of this project was to engineer an unstable cloning vector suitable for easy monitoring of segregation functions in a.
Antibiotic resistance plasmids spread among natural isolates. Broad host range plasmids fems microbiology letters. Broad host range plasmids summary broad host range plasmids are model systems for exploringreplicationmechanismsindiversebacterial species and can extend our. Four new antibioticresistant derivatives of the broadhostrange bhr cloning vector pbbr1mcs have been constructed. Here, we introduce a series of new rk2 based broad host range expression plasmids for inducible production of affinity and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of gram negative hosts which are designed to match the recently suggested modular standard european vector architecture and database. Rocha,1,2 erick denamur4,5 and catherine branger3,4,5 correspondence catherine branger catherine. Finally, the level 2 cloning vector pogg216 rk2based broadhost range, medium copy number was used to construct a dual reporter. A new and improved hostindependent plasmid system for rk2. Broadhostrange plasmid vectors for gene expression in bacteria article pdf available in methods in molecular biology clifton, n.
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. Kovach me, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm. The main aim of this project was to engineer an unstable cloning vector suitable for easy monitoring of segregation functions in a wide range of bacteria. The sulfonamide resistance gene sulii contains psti and hincii sites. Improved determination of plasmid copy number using. Recombinant protein production in escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have an important impact on the success of productivity. It was recognised that a method for accurate and rapid quantification of plasmid copy number is necessary for optimization and better understanding of this. Afterwards, a database search was performed by tandem mass spectrometry ion search algorithms from the mascot house.
Construction of broadhostrange plasmid vectors for easy visible. A plasmid rk2based broad host range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species. The rk2 plasmid is a broadhostrange plasmid belonging to the incp incompatibility group it is notable for its ability to replicate in a wide variety of singlecelled organisms, which makes it suitable as a. Get a printable copy pdf file of the complete article 1. Belongs to a group of vectors atcc 3762237623, 7728777292 differing only in antibiotic resistance and polylinker orientation. 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. These new plasmids have several advantages over many of the currently available. Recombinant protein production in escherichia coli cells is a complex process, where among other parameters, plasmid copy number, structural and segregational stability of plasmid have. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors. Development of broadhostrange vectors and gene banks. The multihost vector prl83a was used in this study. Abstract pbbr1mcs vectors are small in size, contain unique cloning sites within the lacz. This data has been provided by curated databases and other sources that have cited the article.
Macrosiphum euphorbiae both species of aphids are attacked by a large range of natural enemies including lady beetles, flower fly larvae, lacewing larvae. It is designed for use in cyanobacteria, and contains mobilization genes making transfer between bacterial species possible. Rsf 1010 has been completely sequenced scholz 1989. 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. Dec 01, 1995 four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Pdf broadhostrange plasmid vectors for gene expression. Sizereduction of a broad hostrange biobrick cloning vector. Antibiotic resistance plasmids spread among natural. A vector is used to amplify a single molecule of dna into many copes. Broad host range plasmid containing three antibiotic resistance markers. Improvement of pbbr1mcs plasmids, a very useful series of broad host range cloning vectors.
Broadhostrange vector pbhr1 mobilizable boca scientific. Construction of a highly unstable broad host range plasmid. Antibiotic resistance plasmids spread among natural isolates of escherichia coli in spite of crispr elements marie touchon,1,2 sophie charpentier,3,4,5 dominique pognard,3 bertrand picard,4,6 guillaume arlet,7,8 eduardo p. The fosmids used in the conjugation experiments are from a previous study and were constructed using the broad host range cloning vector prs44. Potentiation of curing by a broadhostrange selftransmissible vector for. Replication origin of the broad host range plasmid rk2. 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. The rk2 plasmid is a broad host range plasmid belonging to the incp incompatibility group it is notable for its ability to replicate in a wide variety of singlecelled organisms, which makes it suitable as a genetic engineering tool. Pdf broadhostrange plasmid vectors for gene expression in. Antibiotic resistance plasmids spread among natural isolates of escherichia coli in spite of crispr elements marie touchon,1,2 sophie charpentier,3,4,5 dominique pognard,3 bertrand picard,4,6.
Broad host rangeli plasmid vector pls88 incomplete. A bacterial expression vector archive beva for flexible. Four new derivatives of the broadhostrange cloning vector. Sizereduction of a broad host range biobrick cloning vector. Concentration of induced gene product may be regulated by varying the lac inducer concentration. A new and improved hostindependent plasmid system for rk2based conjugal transfer. Cloning vectors cloning vectors are dna molecules that are used to transport cloned sequences between biological hosts and the test tube.
Host range host range may be wide or narrow broad host. Next generation soil metagenomics largeinsert bac libraries for the discovery of natural products and drugs abstract mark liles2, david mead 1, xing cong li3, kavita kakirde2, rosa ye1, megan wagner. Sonja obranic, fedora babic, gordana maravicvlahovicek pmid 23583732. 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. A versatile onestep crisprcas9 based approach to plasmidcuring. Improvement of pbbr1mcs plasmids, a very useful series of broadhostrange cloning vectors article in plasmid 702 april 20 with 729 reads how we measure reads. Broadhost range plasmids summary broadhostrange plasmids are model systems for exploringreplicationmechanismsindiversebacterial species and can extend our. 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. An autoregulated highlevel expression vector utilizing the tac promoter, rrnb terminators and laciq for lac repression. The main aim of this project was to engineer an unstable cloning vector suitable. In this study, a small broad host range 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.
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. Sizereduction of a broad host range biobrick cloning vector andreas constantinou degree project in biology, bachelor of science, 2010 examensarbete i biologi 15 hp till kandidatexamen, 2010. Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. Kovach me1, elzer ph, hill ds, robertson gt, farris ma, roop rm 2nd, peterson km. Potentiation of curing by a broadhostrange selftransmissible. A gene cloning vector should have capability of autonomous replication, small size, origi. Broad host rangeli plasmid vector pmmb67eh complete. Four new antibioticresistant derivatives of the broad host range bhr cloning vector pbbr1mcs have been constructed. Broadhostrange bacterial cloning vector with the puc1819 mcs and a chloramphenicol resistance gene. Four new derivatives of the broad host range cloning vector pbbr1mcs, carrying different antibioticresistance cassettes.
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