Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Department of Biochemistry and Biophysics, University of California-San Francisco, San Francisco, California, USA.
Nat Chem Biol. 2014 Feb;10(2):99-105. doi: 10.1038/nchembio.1411. Epub 2013 Dec 8.
Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10(54) circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.
遗传电路基于细胞内自由扩散分子之间的相互作用执行计算操作。当转录因子被组合来构建一个电路时，意外的相互作用可能会破坏其功能。在这里，我们应用“部分挖掘”技术从原核基因组中构建了一个包含 73 个 TetR 家族抑制剂的文库。使用体外方法确定了亚组的操纵子，并且利用该信息构建了合成启动子。对启动子和抑制剂进行了交叉反应的筛选。其中，有 16 个被鉴定为既强烈抑制其同源启动子（5 到 207 倍），又与其他启动子表现出最小相互作用。每个抑制剂-启动子对都被转化为 NOT 门并进行了表征。作为一组 16 个 NOT/NOR 门，通过改变输入和输出启动子的模式，可以构建出超过 10(54)个电路。这代表了一组可用于构建用户定义电路的兼容门。
