Building libraries of GFP-tagged neuronal proteins

描述（由申请人提供）：这是一份R21提案，旨在开发和测试一项技术，该技术可能对神经系统新探针的开发产生重大影响，并可能成为我们对基因组的了解和我们对其编码基因的了解之间的桥梁。我们已经创造了一个Tn5转座子，它将绿色荧光蛋白随机插入蛋白质中，产生荧光的三杂交融合蛋白。我们对三种蛋白质的测试表明，如果插入在正确的方向和阅读框中，几乎所有转置插入到编码序列中都会产生荧光蛋白。此外，六分之一的这些蛋白质继续发挥作用。我们的第一个目标是改进这个工具，并对其进行更严格的测试。这将涉及用不同颜色的荧光蛋白以两种方式定位重建转座子。1)将测试相反方向（双头排列），以产生一种转座子，该转座子无论落在哪个方向上都能产生荧光蛋白。这些改进将使在任何一个屏幕上可以回收的荧光蛋白数量增加一倍。如果成功，该工具将使该领域能够更快地开发出基因可解码的活动传感器，以及可用于活体神经元动力学测量的优化荧光能量转移对。该工具将在形成异聚物的两对蛋白质上进行测试，看看它是否能产生荧光能量转移（FRET）对。该提议的第二个目标是生成一个以头对尾结构携带CFP和YFP的转座子。这可能是一个FRET磁带，可以用来扫描一个蛋白质的区域移动激活。这个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，并且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.