According to the known sequence of iron stress-induced gene (isiAB operon), wecloned its 1. 5 kb fragment by PCR, and used this Fragment as integration homologous fragment. Afterseveral steps of subcloning donor DNA i...According to the known sequence of iron stress-induced gene (isiAB operon), wecloned its 1. 5 kb fragment by PCR, and used this Fragment as integration homologous fragment. Afterseveral steps of subcloning donor DNA into the isiAB fragment, a donor plasmid pZL which could be inte-grated into the chromosomal DNA of Synechococcus sp. PCC7942 was constructed. In order to express theheterologous gene at a high level through the integration platform system, we constructed the donor DNAby the following steps. We cloned the strong promoter (240 bp) of heat shock gene groESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS), rbcS polyA intothe downstream of the groESL promoter. The kanamycin resistance gene, as the marker gene, was alsosubcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment andseveral expression elements, such as groESL promoter, MCS, rbcS polyA teminator and kanamycin re-sistance gene, were all included.After naturally transformed and introduced the donor plasmid pZL into Synechococcus sp. PCC7942,as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homolo-gous to the isiAB fragment of Synechococcus sp. PCC7942, the homologous DNA can recombine with thechromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologousDNA were selected. The efficiency of transformation is about 1×10-6. By southem blot analysis, it wasconfirmed that the donor DNA had been integrated into the chromosomal DNA of Synechococcus sp.PCC7942, located on the site of the isiAB gene, and can be replicated with the chromosomal DNA.展开更多
文摘According to the known sequence of iron stress-induced gene (isiAB operon), wecloned its 1. 5 kb fragment by PCR, and used this Fragment as integration homologous fragment. Afterseveral steps of subcloning donor DNA into the isiAB fragment, a donor plasmid pZL which could be inte-grated into the chromosomal DNA of Synechococcus sp. PCC7942 was constructed. In order to express theheterologous gene at a high level through the integration platform system, we constructed the donor DNAby the following steps. We cloned the strong promoter (240 bp) of heat shock gene groESL operon fromSynechococcus sp. PCC7942 by PCR. Then subcloned the multiple cloning sites (MCS), rbcS polyA intothe downstream of the groESL promoter. The kanamycin resistance gene, as the marker gene, was alsosubcloned into the donor DNA. Thus, in the donor plasmid pZL, the integration homologous fragment andseveral expression elements, such as groESL promoter, MCS, rbcS polyA teminator and kanamycin re-sistance gene, were all included.After naturally transformed and introduced the donor plasmid pZL into Synechococcus sp. PCC7942,as in the pZL, the donor DNA sequence is flanked by two DNA fragments (0.4 kb and 0.7 kb) homolo-gous to the isiAB fragment of Synechococcus sp. PCC7942, the homologous DNA can recombine with thechromosomal DNA. After screening by kanamycin, the transformants which integrated the heterologousDNA were selected. The efficiency of transformation is about 1×10-6. By southem blot analysis, it wasconfirmed that the donor DNA had been integrated into the chromosomal DNA of Synechococcus sp.PCC7942, located on the site of the isiAB gene, and can be replicated with the chromosomal DNA.
