Periodic Reporting for period 1 - POLAR (Synthetic Mimicry of Cellular Polarisation)
Période du rapport: 2017-09-01 au 2019-08-31
Some theoretical models suggest that cellular polarisation can spontaneously occur through a simple mechanism that requires: (i) reversible lipid membrane binding, (ii) diffusion while bound upon the membrane, and (iii) positive feedback, i.e. membrane-bound molecules recruit more molecules to bind the membrane. The goal of this project was to test these models by building a synthetic system from the bottom up that recapitulated these key features. As well as helping us understand the mechanism by which symmetry breaking, such as in cellular polarisation, occurs, achieving this goal would also provide useful tools for the creation of ‘proto-cells’—artificial, self-contained systems that exhibit life-like processes. Our strategy incorporated a key tenet of synthetic biology, namely that the system should be built in a modular manner from minimal components. In this case, the minimal modules were to be minimal peptides and protein domains. The project contained two planned milestones: first, to construct a reversible membrane-binding switch; and second, to subsequently add additional modules to generate positive feedback, and thereby generate symmetry breaking.
We explored two designs for membrane targeting: (i) where one binding partner (the ‘anchor’) is permanently attached to the lipid membrane, with a ‘cargo’ molecule that reversible attaches to the anchor from solution via the minimal interaction module, and (ii) where both binding partners are free in solution and membrane binding is switched through changes in their oligomeric state, with monomers remaining in solution and heterodimers binding the membrane. We produced proteins formed from combinations of our minimal modules and tested them on model lipid membranes, successfully achieving both modes of membrane targeting. To achieve the second mode of binding, we had to seek alternative membrane-binding modules and carried out several rounds of design and optimisation to develop a suitable peptide motif.
Due to the additional stages of alteration and refinement required to implement our designs, we had little time to pursue the second milestone of the project, to incorporate additional modules for generating positive feedback. In testing our initial designs we identified several challenges that will need to be circumvented through further engineering cycles as described above for our other modules.