Programmable evolution of optogenetic systems

项目概述：利用光来控制生物过程的能力已经使仔细的 生物学的解剖，并可能发展到未来的治疗失明，癌症，和许多 其它疾病响应于红光到近红外（NIR）光的系统特别有用，因为 这些波长有效地穿透组织，但是这些系统可以通过以下方式大大改进： 将它们工程化以有效结合内源性发色团或对光更敏感。通过 利用一个新的连续进化系统OrthoRep，我们将进化光敏色素B（PhyB） 使用胆绿素（存在于每个细胞中）作为发色团的光遗传学工具， 它们被不同波长的近红外光激活。这将通过修改 自动持续进化（ACE），可以自动施加进化压力， 光遗传学工具的持续突变。因为PhyB会对光线做出反应， 对于生存至关重要，将有可能进化激活波长，即 这是激活基因所需要的，以及一旦被照亮的激活水平。进化的PhyB突变体 具有有用特性的基因将被生物化学验证/表征，并用于控制基因， 哺乳动物细胞由此产生的光遗传学工具将为体内光遗传学提供新的机会 并且修饰的ACE可用于开发许多其它类型的光遗传学工具，光学传感器， 和更多.

Project Summary: The ability to use light to control biological processes has enabled the careful dissection of biology and could be developed into future treatments for blindness, cancer, and many other diseases. Systems that respond to red to near-infrared (NIR) light are particularly useful because those wavelengths efficiently penetrate tissue, but these systems can be greatly improved by engineering them to efficiently bind endogenous chromophores or to be more sensitive to light. By using a new continuous evolution system, OrthoRep, we will evolve the Phytochrome B (PhyB) optogenetic tool to use biliverdin (which is present in every cell) as a chromophore and evolve versions that are activated by different wavelengths of NIR light. This will be accomplished by modifying the Automated Continuous Evolution (ACE) that can automatically apply evolutionary pressure as the gene of the optogenetic tool continuously mutates. Because PhyB will respond to light by activating a gene essential for survival, it will be possible to evolve the activation wavelength, the amount of light that is required to activate the gene, and the level of the activation once illuminated. Evolved PhyB mutants that have useful properties will be validated/characterized biochemically and for controlling genes in mammalian cells. The resulting optogenetic tools will enable new opportunities for in vivo optogenetics and the modified ACE can be used to develop many other types of optogenetic tools, optical sensors, and more.