Biophotonics: Directed Evolution of GFP-based Probes for Glucose

申请人提出使用基于绿色荧光蛋白（GFP）的定向进化方法开发葡萄糖生物传感器。 将编码葡萄糖结合基序的DNA序列文库插入GFP基因中的适当位置，并应用定向进化方法来产生生物探针，该生物探针在与葡萄糖结合时比在未结合状态下发出更亮的荧光。 将使用高分辨率CCD相机对小菌落的荧光进行高通量筛选。 然后使用基于光刻的新方法进行单个小菌落的选择，其中使用数字光处理器将UV光以高分辨率成像到细菌平板上（图案化细胞生长）。 将培养剩余的活细菌，分离质粒，并使用易错PCR和DNA改组的组合启动后续轮的定向进化，以在基因序列中产生额外的变异。 在定向进化的后期阶段筛选菌落也将利用使用扫描共聚焦显微镜和锁模激光的荧光寿命成像。 这将有助于区分葡萄糖结合常数的变化和结合后GFP荧光寿命的变化。 以这种方式产生的基于GFP的探针的优点在于它们可以在大肠杆菌中表达。大肠杆菌中，纯化并直接用于生物传感器应用而无需修饰，或者它们可以在修饰的宿主细胞中表达，产生不断使传感器分子变性的活生物传感器。 这种用于产生基于GFP的生物探针的程序是通用的，并且可以应用于大量潜在的靶分子。

0086920WoodburyThe applicants proposed to develop a glucose biosensor using a directed evolution approach based on the green fluorescent protein (GFP). A library of DNA sequences encoding glucose binding motifs will be inserted into an appropriate position in the gene for GFP and directed evolution methods will be applied to generate a bioprobe that fluoresces much more brightly when bound to glucose than in the unbound state. High throughput screening of fluorescence from microcolonies will be performed using a high resolution CCD camera. The selection of individual microcolonies will then be performed using a novel photolithography based approach in which a digital light processor is used to image UV light onto bacterial plates with high resolution (patterned cell growth). The remaining live bacteria will be grown, plasmids isolated and subsequent rounds of directed evolution will be initiated using a combination of error prone PCR and DNA shuffling to generate additional variation in the gene sequence. Screening of colonies in later stages of the directed evolution will also take advantage of fluorescence lifetime imaging using a scanning confocal microscope and a mode-locked laser. This will help to discriminate between variations in the glucose binding constant and changes in the GFP fluorescence lifetime upon binding. GFP-based probes generated in this way have the advantage that they can either be expressed in E. coli, purified and used directly for biosensor applications without modification, or they can be expressed in modified host cells generating a living biosensor that constantly replenishes the sensor molecule. This procedure for generating GFP-based bioprobes is general and can be applied to a large number of potential target molecules.