A Synthetic Biology Platform for Polarized Protein Scaffold in Escherichia coli
Date Issued
2015
Date
2015
Author(s)
Liu, Yang
Abstract
In the long history of biological evolution, the evolution of unicellular to multicellular is a milestone event. Undoubtedly, the cornerstone of this event is the emergence of cell asymmetry and cell differentiation. The asymmetry of cells gives cell capacity to divide functional areas at the single cell level, even more, to promote the development process of differentiation behavior. Just today, as we trying to reveal the truths of natural philosophy include above themes, synthetic biology is developing rapidly. Synthetic biology is an increasingly sophisticated emerging discipline that dedicates to study life science by constructing and designing artificial genes circuits. On the one hand, synthetic biologists trying to research the natural essence of life by utilizing synthetic parts, circuits, devices and systems. On the other hand, we also research and split natural gene systems to some replaceable standardized DNA parts, and then, use these parts to design and build unnatural life system with desired functions. In the vision of synthetic biology, building asymmetric device in simple symmetric cell has great significance for construction of multi-cellular system from scratch, researching basic mechanisms of cell differentiation and refinement of artificial function in prokaryotes. In our research, we designed and built asymmetric protein scaffold in the most popular and the simplest single-cell gene engineering platform, E. coli. Also, we verify the practicality and robustness of this scaffold system. We first standardized the self-organizing scaffold proteins PopZ and SpmX from Caulobacter crescentus. Then we prove the existence of the direct interaction between them and test the polarized regularity in different expression proportion of these two genes. Based on our research results, we propose a complete theoretical hypothesis ‘Stickiness hypothesis’ to reveal the rule of the interaction between the two proteins. Based on hypothesis, we make a inducible unipolar-bipolar switch (IUBS) scaffold in E. coli. To prove the availability of this scaffold system, we confirmed that the PopZ can regulate the activity of split EYFP fused with SpmX∆C adapter by a Bimolecular Fluorescence Complementation (BiFC) experiment. Even more, the fusion protein includes N terminus of cI repressor and PopZ can change the expression behavior of the downstream gene circuit. As a scaffold molecule, PopZ can affect the activity of transcription factor. Finally, we prove the universality of SpmX∆C as an adapter then make a Micron Cell-based Photovoltaic Unit (MCPU) to show the powerful feature and versatility of this asymmetry platform.
Subjects
Synthetic Biology
Cell Polarization
Protein Scaffold
Bacteriorhodopsin
SDGs
Type
thesis
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