[Construction of recombinate luminescence bacteria vector to evaluate the genetoxic of environment pollutant]

Recombinate luminescence bacteria have the important role in evaluating water toxicity. Two recombinate luminescence bacteria vectors PUCD-uvrA and PUCD-alkA were constructed to investigate the impaired mechanism of pollutant genetic toxicity. The genes of uvrA and alkA were amplified by PCR from E. coli W3110, sequenced after ligated with pGEM-T easy vector. The PCR products and PUCD615 vector were all digested with BamH I, EcoR I, then be linked and imported into JM109 with electrotransformation. Several clones were selected and identificated by PCR and sequencing. The results reviewed that the length of the uvrA and the alkA fragments were 237 bp, 326 bp. When they were sequenced and blasted in GenBank, the homology of sequences reached 99% indicated the amplified results correct. The results of sequencing ligated with PUCD615 reviewed that the fragments of uvrA and alkA had been inserted into the multiple clone site correctly, the insert direction and reading frame were also exactly. Optimizing the condition of ligation and transformation, the large fragment of PUCD615 and the short inserted sequences can been ligated successfully.