2015.1.30文献汇报概要1.ppt

Background Reporter genes are valuable tools for promoter analysis [1],imaging of gene expression [2,3], detecting xenobiotic compounds[4], protein subcellular localization [5], protein-protein interactions[6], and discovery of genes as potential targets for disease [7].Although the current reporter gene assay systems, such as GFP,firefly luciferase (Fluc), LacZ, CAT and GUS have greatly advanced molecular biology research, all except the bacterial luciferase system require additional manipulations such as cell lysis, substrate addition or UV excitation. A distinct advantage of the bacterial luciferase (lux) system is the ability to utilize substrates readily available in the cell (FMNH2, O2, and a fatty aldehyde), thus eliminating the need for cell lysis or exogenous substrate addition. Unfortunately, despite its success as a bacterial reporter, development of the bacterial luciferase as a reporter for widespread use,allowing real-time detection in eukaryotic cells, has faced difficulties, such as low expression levels, lack of thermostability,and poor quantum yield. These hurdles were partially overcome through codon-optimization, addition of specialized linker regions,and co-expression of the flavin reductase gene (frp) encoding the NADPH-FMN oxidoreductase [13], but substantial light production by the lux reaction was demonstrated to occur only in the lower eukaryote Saccharomyces cerevisiae. Materials and Methods 1.Bacterial strains and vectors 2.Construction of plasmids 3.Overexpression and purification of recombinant proteins 4.Random mutagenesis To increase the luciferase activity of luxAB, random mutations were introduced with either three rounds of error-prone PCR or two rounds of chemical mutagenesis. For a typical reaction, 0.2 ng template DNA were added to 100 ml of the error prone PCR system (0.2 mM of each dATP and dGTP, 1.0 mM of each dCTP and dTTP, 7 mM MgCl2, 50 mM KCl, 1 mM MnCl2, 10 mM Tris-HCl (pH 8.3), 0.3 mM of primer luxA-XbaIF and lu