System design of artificial genetic circuit by integration of dry and wet experiment

利用λ噬菌体启动子构建振荡遗传回路。从初步实验来看，pRM启动子的表达弱是引起振荡的一个问题。为了提高pRM启动子的表达水平，我们将几种翻译效率较高的pRM和RBS突变应用到该电路中。尽管做了这些努力，我们还是没有得到连续的振荡。利用lac和tet阻遏因子，我们尝试构建一个拨动开关。将placIq-lacI基因分别导入基因组、低拷贝质粒和中拷贝质粒。用pLlacIq-lacI基因转染高拷贝质粒上的开关。在GFP表达实验中，只有以低拷贝质粒作为lac抑制源的菌株才能达到我们所期望的效果。建立了数学模型。

By using lambda phage promoters, construction of oscillatory genetic circuit was carried out. From the preliminary experiment, the expressional weakness of pRM promoter is problem for the oscillation. In order to increase the expression level from pRM promoter, several mutations of pRM and RBS with higher translation efficiency were applied to this circuit. Despite these efforts, we could not get the continuous oscillation.By using lac and tet repressors, we tried to construct a toggle switch. placIq-lacI gene was introduced into genome, on low copy plasmid, or medium copy plasmid. The toggle switch on high copy plasmid was transformed with each pLlacIq-lacI gene. As the result of the GFP expression experiment, only the strain with low copy plasmid as a source of lac repressor worked as we desired. Mathematical model was also constructed.