Construction Of Genome BAC Library Of Pseudomonas Pseudoalkaligenus YS1

A BAG library was constructed in Escherichia coli from genomic DNA of strain Pseudomonas pseudoalkaligenus YSI, in order to cloning it's. Polyhydroxyalkanoates (PHAs) synthase gene and corresponding regulation gene.Plasmids pBeloBACll and K coli DH10B were employed as vector and host to construct the library of Pseudomonas pseudoalkaligenus YSI. High molecular weight DNA of P. pseudoalkaligenus YSI was prepared by Pulsed Field Gel Electrophoresis (PFGE), and ligated with the purified and dephosphorated vector DNA. The ligation was then electroporated into competent cell, and cultured on agar plates containing chloramphenicol, X-gal and IPTG for screening positive colonies. White colonies were picked out and preserved on agar plates.Plasmids of the positive colonies were extracted, digested by Notl enzyme, and detected with agarose electrophoresis. Two DNA fragments appeared, one fragment is 7.5 kb and another is more than 23 kb. The 23 kb fragment should be the foreign fragment. The colony hybridization was employed to confirm the existence of foreign fragment. Ninety-five percent of the library showed positive results, and the negative contrast didn't show hybridization sign. The BAG library consists of 1000 clones, provides more than 4.8-fold coverage of the P. pseudoalkaligenus YSI genome.In order to obtain some probes for further PHA synthase gene cloning from the library, RAPD methods was employed to screen for some random primers for this purpose. The amino acid sequences of two primers coded are the same as or very similar to the conservative sequences of PHAs synthase of ten strains employed. These two primers or their amplify fragments may be used as probes in further investigation.