Siegal-Gaskins Dan, Tuza Zoltan A, Kim Jongmin, Noireaux Vincent, Murray Richard M
Division of Biology and Biological Engineering, California Institute of Technology , Pasadena, California 91125, United States.
ACS Synth Biol. 2014 Jun 20;3(6):416-25. doi: 10.1021/sb400203p. Epub 2014 Apr 7.
The many successes of synthetic biology have come in a manner largely different from those in other engineering disciplines; in particular, without well-characterized and simplified prototyping environments to play a role analogous to wind-tunnels in aerodynamics and breadboards in electrical engineering. However, as the complexity of synthetic circuits increases, the benefits--in cost savings and design cycle time--of a more traditional engineering approach can be significant. We have recently developed an in vitro "breadboard" prototyping platform based on E. coli cell extract that allows biocircuits to operate in an environment considerably simpler than, but functionally similar to, in vivo. The simplicity of this system makes it a promising tool for rapid biocircuit design and testing, as well as for probing fundamental aspects of gene circuit operation normally masked by cellular complexity. In this work, we characterize the cell-free breadboard using real-time and simultaneous measurements of transcriptional and translational activities of a small set of reporter genes and a transcriptional activation cascade. We determine the effects of promoter strength, gene concentration, and nucleoside triphosphate concentration on biocircuit properties, and we isolate the specific contributions of essential biomolecular resources-core RNA polymerase and ribosomes-to overall performance. Importantly, we show how limits on resources, particularly those involved in translation, are manifested as reduced expression in the presence of orthogonal genes that serve as additional loads on the system.
合成生物学的诸多成功之处，其实现方式与其他工程学科大不相同；尤其是，没有具备充分特征且简化的原型制作环境来发挥类似于空气动力学中的风洞以及电气工程中的面包板的作用。然而，随着合成电路复杂性的增加，采用更传统工程方法在节省成本和缩短设计周期方面的益处可能会非常显著。我们最近基于大肠杆菌细胞提取物开发了一种体外“面包板”原型制作平台，该平台能让生物电路在比体内环境简单得多但功能相似的环境中运行。这个系统的简单性使其成为快速生物电路设计与测试以及探究通常被细胞复杂性所掩盖的基因电路运作基本方面的一个很有前景的工具。在这项工作中，我们通过对一小部分报告基因的转录和翻译活动以及一个转录激活级联进行实时同步测量来表征无细胞面包板。我们确定了启动子强度、基因浓度和三磷酸核苷浓度对生物电路特性的影响，并分离出关键生物分子资源——核心RNA聚合酶和核糖体——对整体性能的具体贡献。重要的是，我们展示了资源限制，特别是那些与翻译相关的限制，是如何在作为系统额外负载的正交基因存在时表现为表达降低的。
