+ Translate
+ Most Popular
Investigations on the organic drift in North Swedish streams
Limitations of Using Microsoft Excel Version 2016 (MS Excel 2016) for Statistical Analysis for Medical Research
Phloem necrosis of coffee in Surinam
Anatomy of Mystus seenghala IV Nervous system
Integrated farming system for gardenland conditions of Coimbatore district - an over view
Tetrapleure (Tetrapleura tetraptera), an unknown African medicinal and spice plant
The origin and phylogenetic significance of the trochophoran larvae 2. evolutionary significance of the larvae of coelomate worms and mollusks
Comparison of rice bran and maize bran as feeds for growing and fattening pigs
Enterobacter amnigenus. An unusual human pathogen
Influence of Seriboost foliar application on leaf yield and leaf protein content in mulberry (Morus spp.), in relation to silkworm cocoon production
The identity of the lipstick mold of cultivated mushrooms agaricus bisporus
Advantages and disadvantages of bordeaux mixture and of lime-sulphur used on apples in the growing season
'Pan-sukh' disease of Rice in the Central Provinces
Geological age of the Ptilophyllum flora; a critical reassessment
Study of vitellogenesis in birds; physiological phases & role of folliculin in vitellogenesis
Evaluation of WCT coconut and Komadan coconut
Therapy for acne with saccharomyces boulardii
Evidence for Late Cretaceous N-S dextral shear in the west-central crystalline core, North Cascades, Washington
'Rajeshwari' - a high-yielding white seeded variety of sesame for Andhra Pradesh
Manufacture of Ricotta cheese from whey fortified with skim milk powder using different acidulants
Occurrence of Eutrichophilus mexicanus (Rudow, 1866) and Eutrichophilus lobatus (Ewing, 1936) (Phthiraptera: Trichodectidae) on Sphiggurus villosus (Cuvier, 1825) (Rodentia: Erethizontidae) in Rio de
Factors affecting fruitfulness in durian (Durio zibethinus Murr.). I. Flowering and pollination
Bronchial cuff pressure change caused by left-sided double-lumen endobronchial tube displacement
Some Biological Applications of Organometallic Compounds
The composition of pampas-grass (Cortaderia argentea.)

Blue/white screening of recombinant plasmids in gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression

Blue/white screening of recombinant plasmids in gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression

Gene 219(1-2): 91-99

ISSN/ISBN: 0378-1119

PMID: 9757005

DOI: 10.1016/s0378-1119(98)00396-5

The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ. Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 008238482

Download citation: RISBibTeXText

Related references

Evaluation of the Staphylococcus aureus class C nonspecific acid phosphatase (SapS) as a reporter for gene expression and protein secretion in gram-negative and gram-positive bacteria. Applied and Environmental Microbiology 73(22): 7232-7239, 2007

Expression of a chloramphenicol-resistance determinant carried on hybrid plasmids in gram-positive and gram-negative bacteria. Gene 32(1-2): 151-160, 1984

Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein. Molekuliarnaia Biologiia 37(6): 989-998, 2003

Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene. Journal of Bacteriology 164(3): 1376-1380, 1985

Mucosal delivery of microbicides by recombinant commensal bacteria Expression of the HIV-inactivating protein cyanovirin-N in gram-positive bacteria. AIDS 15(Suppl. 1): S58, 2001

Rolling circle-replicating plasmids from gram-positive and gram-negative bacteria: a wall falls. Molecular Microbiology 8(5): 789-796, 1993

A diplotype in the lymphotoxin alpha gene is associated with differential expression of LTA mRNA induced by Gram-positive and Gram-negative bacteria. International Journal of Immunogenetics 34(3): 157-160, 2007

Enterobacterial gene expression of antibiotic resistance under the control of Bacillus thuringiensis regulatory signals in gram-negative and gram-positive bacteria. Genetika 18(11): 1825-1834, 1982

A Dual-Replicon Shuttle Vector System for Heterologous Gene Expression in a Broad Range of Gram-Positive and Gram-Negative Bacteria. Current Microbiology 75(10): 1391-1400, 2018

Expression of an entobacterial gene for antibiotic resistance under the control of regulatory signals of Bacillus thuringiensis in gram-negative and gram-positive bacteria. Soviet genetics(pub 1983) 18(11): 1356-1365, 1983

New system for positive selection of recombinant plasmids and dual expression in yeast and bacteria based on the restriction ribonuclease RegB. Biotechnology Progress 19(3): 727-733, 2003

Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria. Fems Microbiology Reviews 21(4): 337-368, 1998

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria. Bmc Biotechnology 5: 3, 2005

Gene expression in Gram-positive bacteria. Tanpakushitsu Kakusan Koso. Protein Nucleic Acid Enzyme 28(14): 1468-1479, 1983

Gene expression using gram-positive bacteria. Ladisch, M R, Bose, A ACS Conference Proceedings Series; Harnessing biotechnology for the 21st century: 18-22, 1992