Engineering phosphorylation-dependent post-translational protein devices

One goal underlying synthetic biology is to develop standard biological parts that can be reliably assembled into devices encoding higher-order functions. Here, I developed a framework for engineering post-translational devices, which are devices whose inner workings are modulated by non-covalent protein interactions and covalent protein modifications. To test the framework, I designed a scaffold for engineering post-translational devices in yeast, the Phospholocator, that can be used to assemble peptide parts in order to produce devices that couple upstream kinase activity to regulated nuclear translocation. I used the Phospholocator to design, build, and characterize a Phospholocator device, the Cdc28-Phospholocator, whose location is regulated by the activity of cyclin-dependent kinase Cdc28. I next engineered and tested a Fus3-Phospholocator device, whose location is regulated by the activity of the mitogen-activated protein kinase Fus3, in order to demonstrate that the Phospholocator scaffold supports the engineering of many post-translational devices. I used the Cdc28-Phospholocator to follow Cdc28 activity levels throughout the yeast cell cycle, thereby illustrating the utility of the Cdc28-Phospholocator as a tool for biological inquiry. To implement more complex functions, device engineers will want to connect post-translational devices to build multi-component systems. I thus developed a model for device composition that features a universal signal carrier that is both input into and output from post-translational devices. The universal signal could enable engineers to easily combine devices in any desired order, and thus build many new post-translational systems.

Department dc:contributor.department

Massachusetts Institute of Technology. Biological Engineering Division.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Sutton, Samantha C. (Samantha Carol)

Advisor dc:contributor.advisor

• Drew Endy.

• Biological Engineering Division.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/45205

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/45205