Design and construction of a synthetic evolutionary circuit based on tryptophan sensing in Escherich

来源 :2015合成生物学青年学者论坛 | 被引量 : 0次 | 上传用户:ydzdems
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  Combining metabolites sensing based high-throughput screening with the strategy of adaptive laboratory evolution, the thesis proposed a concept called synthetic evolutionary engineering (SEE) for the construction of microbial cell factory.The idea behind SEE is to connect microbe's fitness in a selective pressure with the intracellular titer of a specific metabolite, and then use the strategy of adaptive laboratory evolution to select for high producers of the targeted metabolite which fit the selective environmental condition best.Major conclusions are listed below:(1) Proposed the survival pressure strategy based on carbon source utilization.Using maltose as selective pressure, designed a genetic circuit called survival pressure circuit which expresses maltase induced by tryptophan, connecting microbe's fitness with its tryptophan productivity.Meanwhile, established a model to describe the transfer function of the genetic circuit, predicting that microbe's fitness increase first and then decrease with the increasing tryptophan productivity.(2) Constructed the genetic circuit which expresses maltase induced by tryptophan.The expression level of reporter gene sfGFP positively responses to the concentration of tryptophan added externally in a range of 0mM to 0.6mM.(3) The growth under M9 maltose medium of the E.coli, which transferred with the synthetic genetic circuit, positively responses to the concentration of tryptophan added externally in a range of 0mM to 0.05mM, showing the survival strategy to be effective.Based on the transfer function of the genetic circuit, proposed a model of evolution, predicting that the synthetic genetic circuit can effectively select for high producers of tryptophan in a mixed bacterial culture, and in a limited number of passage, realizing the process of self-enrichment of high tryptophan producers.The thesis proposed the idea of synthetic evolutionary engineering for the construction of microbial cell factory for the first time, and successfully connect microbe's fitness in a selective pressure with the intracellular titer of a specific metabolite using the survival pressure circuit, providing key support for the evolution of high producers using survival pressure strategy.
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