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ABSTRACT: A genomic library of Thermus sp. FD3041 which produces thermostable alkaline phosphatase (FD-TAP) was constructed with the vector pUC118 and the host E. coli TG1. 3-10kb inserted fragments of foreign DNA were identified in 90 percent of the 12,000 clones thus obtained. Five positive clones were detected after screening the plated library by the method of colony coloration for TAP in situ. Preliminary analysis of the enzyme expressed from one recombinant plasmid pTAP362 showed that the properties of the recombinant enzyme, such as the thermal stability and optimal temperature of reaction, were identical to those of the native enzyme. The gene of FD-TAP was located on the 2.0kb BamHI-HindIII fragment of the pTAP362, determined by its physical map and the change of enzyme activity in different partially deleted plasmids. Results of thermostability experiment in PCR thermal cycle showed that the FD-TAP would be suitable for labelling of primers and detection of PCR amplified products.
Shanghai, Shanghai Shi, China
- Institute of Genetics