vector permitting visual detection of recombinants
Comments
To detect nuclease activity, use TBD agar: 100 ml 0.05 M Tris, pH 9; 1 g agar; 1 g NaCl; 10 ul CaCl2, 1 M; ssDNA (0.5% w/v). Heat until DNA and agar are dissolved and melted. Add 9 mg Toluidine blue O dye (Sigma T3260) and heat again. Melt for use.
Replicates as a rollling circle to a level of 20-30 copies per chromosome in L. lactis.
High copy-number plasmid vector for gram positive bacteria. Permits identification of recombinants by detecting secreted nuclease activity.
Phenotype(s) verified: eryR
Media
ATCC® Medium 1640: LB medium (ATCC medium 1065) with 25 mcg/ml erythromycin
Growth Conditions
Temperature: 30.0°C
References
Le Loir Y, et al. Direct screening of recombinants in gram-positive bacteria using the secreted staphylococcal nuclease as a reporter. J. Bacteriol. 176: 5135-5139, 1994. PubMed: 8051029
Lachica RV, et al. Metachromatic agar diffusion methods for detecting staphyloccal nuclease activity. Appl. Microbiol. 21: 585-587, 1971. PubMed: 4930272
Kok J, et al. Construction of plasmid cloning vectors for lactis streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl. Environ. Microbiol. 48: 726-731, 1984. PubMed: 6095756
