| RRC ID |
33503
|
| 著者 |
Metcalf WW, Jiang W, Daniels LL, Kim SK, Haldimann A, Wanner BL.
|
| タイトル |
Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria.
|
| ジャーナル |
Plasmid
|
| Abstract |
We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi. They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria. Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.
|
| 巻・号 |
35(1)
|
| ページ |
1-13
|
| 公開日 |
1996-1-1
|
| DOI |
10.1006/plas.1996.0001
|
| PII |
S0147-619X(96)90001-3
|
| PMID |
8693022
|
| MeSH |
Alleles
Bacillus subtilis / genetics*
Base Sequence
Cloning, Molecular
Conjugation, Genetic
DNA Replication
DNA, Bacterial / genetics
Escherichia coli / genetics*
Genetic Vectors / genetics*
Molecular Sequence Data
Mutagenesis
beta-Galactosidase / genetics
|
| IF |
2.805
|
| 引用数 |
345
|
|
WOS 分野
|
MICROBIOLOGY
GENETICS & HEREDITY
|
| リソース情報 |
| 原核生物(大腸菌) |
pAH34
pBW30
pLD53
pLD54
pLD55
pWM2
pWM3
pWM4
pWM41
pWM43
pWM45
pWM47
pWM5
pWM6
pWM7
pWM71
pWM73
pWM75
pWM77
pWM91
pWM93
pWM95
pWM97 |
