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 i s 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.
| Sheridan, D L; Robert, A; Cho, C H et al. (2006) Regions of alpha-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid receptor subunits that are permissive for the insertion of green fluorescent protein. Neuroscience 141:837-49 |
| Sheridan, Douglas L; Hughes, Thomas E (2004) A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins. BMC Biotechnol 4:17 |
