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pRE107 plasmid in E. coli

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編號:B172250

品牌:Mingzhoubio

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產品名稱

pRE107 plasmid in E. coli

商品貨號

B172250

Designations

pRE107 plasmid in E. coli

Depositors

DM Schifferli, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA

培養基

Biosafety Level

Host

Escherichia coli SM10 lambda pir

Vector Information

Size (kb): 6.55

Vector: pRE107 (plasmid)

Construction: pGP704, sacB1 (pUC58)

Marker(s): ampR,sacB

Construct size (kb): 6.55

Features: marker(s): ampR
marker(s): sacB1
replicon: conditional R6K ori
replicon: oriT
MCS: PstI...EcoRI
restriction site: EcoRI HindIII

Applications

Counterselection levansucrase
Suicide vector
Vector permitting allelic exchange of cloned inserts

Comments

Restriction digests of the clone give the following sizes (kb): BamHI-- 3.2, 1.7, 1.5; XbaI-- 6.55; EcoRI-- 4.0, 2.8.

The sacB1 allele is a variant of sacB with certain restriction sites removed by site directed mutagenesis. Expression of sacB confers sensitivity to sucrose.

The conditional R6K origin of replication requires that the pi protein be expressed in trans for plasmid maintenance.

One of four allelic exchange suicide vectors (ATCC 87691 - 87694) that provide both selection for chromosomal integration (ampR, cmlR, kanR or tetR) and counterselection for loss of vector DNA and the wild type allele.

Construct is suicide plasmid in any host not expressing pir.

Cloned inserts may be integrated into the host chromosome (a single recombination event) following electroporation and appropriate antibiotic selection.

Negative selection for sucrose sensitivity (sacB) selects for a second recombination event resulting in loss of vector DNA.

Media

ATCC^® Medium 1227: LB Medium (ATCC medium 1065) with 50 mcg/ml ampicillin

Growth Conditions

Temperature: 37°C

References

Chang TW, et al. Immunoassay for biologically active human interferon-gamma employing unique monoclonal antibodies. US Patent 4,666,865 dated May 19 1987

Edwards RA, et al. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207: 149-157, 1998. PubMed: 9511756

Donnenberg MS, Kaper JB. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun. 59: 4310-4317, 1991. PubMed: 1937792

Blomfield IC, et al. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol. Microbiol. 5: 1447-1457, 1991. PubMed: 1686293

Kolter R, et al. Trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid R6K. Cell 15: 1199-1208, 1978. PubMed: 728998

McIver KS, et al. The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol. Microbiol. 18: 877-889, 1995. PubMed: 8825092

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