Biophotonics: Directed Evolution of GFP-based Probes for Glucose

申请人建议使用基于绿色荧光蛋白(GFP)的定向进化方法来开发葡萄糖生物传感器。编码葡萄糖结合基序的DNA序列文库将被插入到GFP基因的适当位置，并将应用定向进化方法来生成生物探针，该探针在与葡萄糖结合时比在未结合状态下发出更明亮的荧光。将使用高分辨率的ccd相机高通量地筛选来自微菌落的荧光。然后，将使用一种基于光刻的新方法进行单个微菌落的选择，在该方法中，使用数字光处理器将紫外线成像到高分辨率的细菌平板上(图案化细胞生长)。剩下的活细菌将被培养，分离出质粒，随后几轮的定向进化将使用容易出错的PCR和DNA改组的组合来启动，以在基因序列中产生额外的变异。在定向进化的后期阶段，对菌落的筛选也将利用扫描共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时GFP荧光寿命的变化。以这种方式产生的基于GFP的探针具有以下优点：它们既可以在大肠杆菌中表达、纯化并直接用于生物传感器，而不需要修饰，也可以在修饰的宿主细胞中表达，产生不断补充传感器分子的活的生物传感器。这种生成基于GFP的生物探针的方法是通用的，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.