| 초록 |
From the beginning, searching for the useful genes in huge library has been focused on two screening methods. These are nucleotide sequence-based screening and enzyme activity-based screening. Before applying these screening methods, FACS has been widely used to select the positive clones in liquid cultures. Here we suggest the photochemical selection method of positive clones using photosensitive mutant of E. coli and vector system. We constructed the light sensitive mutant of E. coli and vector system. The selection system consists of hemH negative E. coli and p19hemH (derivative of pUC19) which contain SD sequence and hemH gene. Without proper promoter or promoter with genes, this mutant simply can not survive under light emission. From the system constructed, only light radiation was required to select positive colonies. Through this simple and comfort method, we could remove the clones containing self-ligation plasmids and selectively obtain positive clones in both liquid and solid cultures. Using library obtained via this method, we are currently screening various useful genes from huge libraries. |
