Abstract：
:An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified φCTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Omega elements), and (6) the φCTX attachment site. The MCS and Omega elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase. In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacI(q) also contained on the plasmid. Upon conjugal transfer, mini-CTX1 and mini-CTX2 integrated at frequencies of 10(-8) and 10(-7), respectively. The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI.
journal_name
Plasmidjournal_title
Plasmidauthors
Hoang TT,Kutchma AJ,Becher A,Schweizer HPdoi
10.1006/plas.1999.1441keywords:
subject
Has Abstractpub_date
2000-01-01 00:00:00pages
59-72issue
1eissn
0147-619Xissn
1095-9890pii
S0147-619X(99)91441-5journal_volume
43pub_type
杂志文章相关文献
PLASMID文献大全abstract：:The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep reg...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2005.08.003
更新日期：2006-03-01 00:00:00
abstract：:As multidrug resistant bacteria pose one of the greatest risks to human health new alternative antibacterial agents are urgently needed. One possible mechanism that can be used as an alternative to traditional antibiotic therapy is transfer of killing agents via conjugation. Our work was aimed at providing a proof of ...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2015.10.001
更新日期：2015-11-01 00:00:00
abstract：:Two B/O plasmids were sequenced. The kanamycin resistance plasmid R805a was found in a Salmonella Typhi strain from a 1972 typhoid fever outbreak in Mexico City. pCERC6, which confers resistance to ampicillin, streptomycin and sulphamethoxazole, was found in a commensal E. coli isolated from a healthy adult in Sydney ...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2019.02.005
更新日期：2019-03-01 00:00:00
abstract：:Plasmid pT181 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, Inc3A and Inc3B. Inc3A incompatibility is expressed by the primary replication control determinant, copA, which specifies two small transcripts, RNA I and R...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(90)90039-f
更新日期：1990-01-01 00:00:00
abstract：:To study the transcriptional activity of the HIV-1 LTR, we constructed a vector containing Renilla and Firefly luciferase genes under the control of the LTR (wild-type or mutated version) and oriented in a manner that allowed them to be transcribed in opposite directions. We found that the HIV-1 LTR acted as a bidirec...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2014.06.001
更新日期：2014-07-01 00:00:00
abstract：:The CoIV plasmids are large virulence plasmids of the incompatibility group IncFI. We have obtained the genetic maps of two of the most studied CoIV plasmids, pCoIV-B188 and pCoIV-K30, according to restriction enzyme sites and landmark phenotypic characteristics such as colicin V, the aerobactin iron uptake system, th...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(89)90007-3
更新日期：1989-11-01 00:00:00
abstract：:A preliminary screening study of six Moraxella catarrhalis isolates from primary school children in the Netherlands identified a small 3.5 kb plasmid (pEMCJH03), containing four open reading frames, which encoded three mobilizing and one replicase protein. Insertion of a kanamycin containing transposon (yielding pEMCJ...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2004.11.003
更新日期：2005-05-01 00:00:00
abstract：:Corynebacterium glutamicum is an industrial microorganism for production of amino acids. However, the metabolic engineering in C. glutamicum has been retarded due to lack of suitable vectors. In this study, we have constructed a shuttle vector pDXW-10 which harbors a large multiple cloning site suitable for cloning mu...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2010.05.004
更新日期：2010-09-01 00:00:00
abstract：:The Gram-positive Corynebacterium glutamicum is widely known for its application in the industrial production of amino acids and as a non-pathogenic model organism for cell wall biosynthesis in the group of CMN bacteria. For biotechnological and physiological studies often co-expression of recombinant genes is require...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2018.01.001
更新日期：2018-01-01 00:00:00
abstract：:Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained wi...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.1996.0036
更新日期：1996-09-01 00:00:00
abstract：:We describe here the construction of Gateway-compatible vectors, pBGP1-DEST and pPICZα-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. Both vectors direct the synthesis of fusion proteins consisting of the N-terminal signal and pro-sequences of...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2010.08.004
更新日期：2011-01-01 00:00:00
abstract：:We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy nu...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(92)90022-3
更新日期：1992-05-01 00:00:00
abstract：:pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthe...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(91)90042-u
更新日期：1991-11-01 00:00:00
abstract：:The conjugative shuttle transposon Tn1545 from Streptococcus pneumoniae confers resistance to kanamycin (aphA-3), erythromycin (ermAM), and tetracycline (tetM). The 25.3-kb element is self-transferable to various gram-positive bacterial genera where it transposes. Tn1545 is also capable of transposition, but not of co...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(87)90009-6
更新日期：1987-01-01 00:00:00
abstract：:Two improved Deinococcus radiodurans-Escherichia coli shuttle vectors have been constructed. pI3 is a 16-kb plasmid that confers chloramphenicol resistance in D. radiodurans (CmR, cat) and ampicillin resistance in E. coli (ApR) and contains a multiple cloning site that does not interrupt sequences necessary for replic...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(92)90057-h
更新日期：1992-11-01 00:00:00
abstract：:A simple and rapid method for determining plasmid copy number is described. The eukaryotic luc gene is used as a marker to tag plasmid derivatives of several well-known vectors, and by measuring light activity plasmid copy number is determined. A comparative analysis using a standard hybridization procedure to estimat...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.1994.1074
更新日期：1994-11-01 00:00:00
abstract：:The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon. So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmi...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/s0147-619x(02)00100-2
更新日期：2002-09-01 00:00:00
abstract：:A circular map of the IncHI plasmid R27 corresponding to a genome size of 182 kb was established using the restriction endonucleases ApaI, XbaI, and PstI. The map was derived from the results obtained by hybridizing individual ApaI and XbaI fragments to blotted digests of the plasmid, as well as from complete and part...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(85)90058-7
更新日期：1985-01-01 00:00:00
abstract：:A simple mathematical model of drug-induced plasmid elimination (curing) considering density-dependent growth rates and plasmid transfers is presented. It describes nonlinear population dynamics of conjugative plasmids during in vitro curing experiments in batch culture. The model was tested on kinetics of acridine or...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(89)90052-8
更新日期：1989-03-01 00:00:00
abstract：:Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two su...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.2001.1537
更新日期：2001-09-01 00:00:00
abstract：:Strain LBG A3136 of Nocardia mediterranei (ETH Collection) was found to contain a low-copy-number covalently closed circular extrachromosomal element, pMEA 100, which could only be isolated from mycelium grown on agar plates. pMEA 100 could not be isolated from the closely related strain ATCC 13685. Hybridization expe...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(85)90072-1
更新日期：1985-09-01 00:00:00
abstract：:Isolates of Rhodococcus equi from pneumonic foals possess an 85- or 90-kb virulence-associated plasmid. A prominent, thermoregulated surface antigen, VapA, encoded by these plasmids is thought to be important in virulence. A 135-kb fragment containing the origin of replication of R. equi strain 103 virulence-associate...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.1997.1311
更新日期：1997-01-01 00:00:00
abstract：:A chimeric plasmid, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7, a large plasmid from Oenococcus oeni, and pEM64, a plasmid derived from pIJ2925 and containing a gene for resistance to chloramphenicol. pRS7Rep is a shuttle vector that replicates in Escherichia coli using its pIJ2925 componen...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2014.11.004
更新日期：2015-01-01 00:00:00
abstract：:Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique Ba...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.2002.1565
更新日期：2002-03-01 00:00:00
abstract：:Plasmid pRK404-a smaller derivative of RK2-is a tetracycline-resistant broad-host-range vector that carries a multiple cloning site and the lacZ(alpha) peptide that enables blue/white selection for cloned inserts in Escherichia coli. We present herein the complete and annotated sequence of pRK404 and three related vec...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/s0147-619x(03)00030-1
更新日期：2003-07-01 00:00:00
abstract：:To facilitate the study of the structure-function relationship of human hemoglobin (Hb A), we have developed a new hemoglobin expression vector, pGEX6P-alpha-[SD]-beta. This vector allows the co-expression of alpha-Hb as a fusion protein with Glutathione S-Transferase (GST-alpha-Hb) and beta-Hb with an additional meth...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2008.09.006
更新日期：2009-01-01 00:00:00
abstract：:An RP4-oriT shuttle vector pJIR1457 originally developed for Clostridium perfringens was successfully transferred by conjugation from Escherichia coli to Clostridium botulinum type A strains and to a nontoxigenic C. botulinum type A-transposon Tn916 mutant strain lacking the entire toxin gene cluster. The light chain ...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.1998.1366
更新日期：1998-11-01 00:00:00
abstract：:Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRM1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the oriT region from pRK2, a...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1006/plas.1995.1024
更新日期：1995-05-01 00:00:00
abstract：:The incompatibility region of the Streptomyces plasmid pSL1 was identified as a 240-bp segment, though some other function from the vector plasmid was also necessary. A 540-bp fragment including the 240-bp region was enough for full expression of incompatibility. Inserted mutation analysis led to a more detailed locat...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/0147-619x(87)90021-7
更新日期：1987-03-01 00:00:00
abstract：:Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the r...
journal_title：Plasmid
pub_type： 杂志文章
doi：10.1016/j.plasmid.2006.07.005
更新日期：2007-01-01 00:00:00
