Building libraries of GFP-tagged neuronal proteins

描述（由申请人提供）：这是一项R21提案，旨在开发和测试一项可能对神经系统新探针的开发产生重大影响的技术，该技术可能作为我们对基因组的了解与我们想了解的基因编码之间的桥梁。 我们已经创建了一个Tn5转座子，将GFP随机插入蛋白质中，产生荧光的三杂交体融合蛋白。 我们对三种蛋白质的测试表明，如果插入的方向和阅读框正确，几乎所有转座插入编码序列都会产生荧光蛋白。此外，这些蛋白质中有六分之一继续发挥作用。 我们的首要目标是改进这个工具，并更严格地测试它。 这将涉及重建转座子与不同颜色的荧光蛋白定位在两种方式。 1)相反的方向（双头排列）将被测试，以创建一个转座子，产生荧光蛋白，而不管它的土地方向。这些改进将使在任何一个屏幕中可以回收的荧光蛋白的数量增加一倍。 如果成功，该工具将使该领域能够更快地开发可遗传解码的活动传感器以及可用于在活神经元中进行动力学测量的优化荧光能量转移对。该工具将在两对形成异源寡聚体的蛋白质上进行测试，看看它是否可以产生荧光能量转移（FRET）对。该提议的第二个目的是产生以头对尾构型携带CFP和YFP的转座子。 这是一个潜在的FRET盒，可以用来扫描蛋白质的区域，移动激活。 该工具将在两种蛋白质上进行测试，我们对它们的结构有很好的了解，并可以控制它们的激活。

DESCRIPTION (provided by applicant): This is an R21 proposal to develop and test a technology that may have a large impact on the development of new probes for the nervous system, and which may serve as a bridge between what we know about the genome and what we want to know about the genes it encodes. We have created a Tn5 transposon that inserts GFP randomly into the proteins, producing tribrid fusion proteins that are fluorescent. Our tests of three proteins show that virtually all of the transposed insertions into coding sequences produce fluorescent proteins if the insertion is in the correct orientation and reading frame. Moreover, 1 in 6 of these proteins continue to function. Our first aim is to improve this tool, and test it more rigorously. This will involve rebuilding the transposon with different colored fluorescent proteins positioned in two ways. 1) Opposing orientations (a double-headed arrangement) will be tested to create a transposon that produces a fluorescent protein regardless of the orientation it lands in. These improvements will double the number of fluorescent proteins that can be recovered in any one screen. If successful, this tool should empower the field to more quickly develop genetically decodable sensors of activity as well as optimized fluorescence energy transfer pairs that can be used to make kinetic measurements in living neurons. This tool will be tested on two pairs of proteins that form heteroligomers to see if it can generate fluorescence energy transfer (FRET) pairs. The second aim of the proposal is to generate a transposon carrying CFP and YFP in a head-to-tail configuration. This is potentially a FRET cassette that could be used to scan a protein for regions that move up on activation. This tool will be tested on two proteins where we have good information about their structure and can control their activation.