Engineering ââplug and playââ biosensors in Escherichia coli

The ability of biological components to function reliably in different genetic contexts is one of the underlying fundamental principles in synthetic biology. Application of this principle allows for the development of distinct parts or bio-bricks and for the possibility of genetic abstraction. However, the sensitivity of genetic elements to changes in context can cause issues for this abstracted view of genetic elements as parts. We look at how this context sensitivity effects DNA and RNA based biosensors and we further investigate the ability of feedback to reduce noise in a biological device. Riboswitches are structural genetic regulatory RNA elements that directly couple the sensing of small molecules to gene expression. They are able to sense a wide range of small molecules and in response regulate gene expression. We investigated how changes in genetic context effected riboswitch function and how these changes affected expression from a riboswitch and adaptation of a riboswitch to a novel downstream application. To overcome these contextual difficulties with riboswitches, we have designed and introduced a novel genetic element called a ribo-attenuator. This genetic element allows for predictable tuning, insulation from contextual changes and a reduction in expression variation. Ribo-attenuators allow riboswitches to be treated as a truly modular and tunable component, and thus increase their reliability for a wide range of applications. Many biological processes use a form of feedback to achieve a highly robust and reliable output. Accurate control of a biological process is essential for critical functions in biology, from the cell cycle to proteome regulation. We design a tunable synthetic feedback network using a class of viral proteins called Integrases, which can flip the orientation of DNA segments in a digital manner. Excisionase feedback closes the loop in this system, creating a novel switch that provides a tunable and modular architecture for applications throughout synthetic biology and bio-manufacturing that require a highly robust and orthogonally controlled output. This system is highly orthogonal and evolutionary robust, and demonstrates a strong capability for regulating and reducing the variability in expression genes being transcribed under its control.