| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 104 === In synthetic biology, there are many techniques to control where protein products are expressed. However, most of these tools only specify a certain target (eg. organelles, membrane or outside of the cell), instead of a spatially defined region. Our study, PopZ-based cellular polarity module, is able to localize cellular components to one or both cell poles, and provide a way to achieve an unprecedented level for spatial regulation of gene expression.
In this system, Caulobacter crescentus proteins are expressed in Escherichia coli to test their applicability in an orthogonal context. PopZ is adopted as the cell pole identifier, while SpmX’s muramidase domain is used as an adapter to bring effector proteins toward the PopZ-tagged pole. We first characterized the distribution of heterologously expressed molecules against four parameters: expression level, oligomerization efficiency, SpmX intactness and dilution rate, and found conditions where PopZ became either bipolar, unipolar or diffused. Furthermore, by using flow cytometry and time-lapse imaging, we demonstrated that under certain conditions, the introduced proteins could be asymmetrically partitioned to the daughter cells.
Next, we showcased three main features of the module: scaffold, polarization and asymmetric cell division. In 2015, Liu showed that PopZ aggregation can act as a scaffold to locally increase the interaction of target proteins using a split yellow fluorescent protein. We further demonstrated that our system can polarize cellular components of interest, using bacteriorhodopsin-based “micron battery” as an example. Moreover, T7 polymerase and mfLon protease were utilized to show that the system can also accomplish asymmetric cell division.
The results of this study present a promising platform on which the synthetic biology community can build more complicated systems. On the other hand, they also provide useful insights to asymmetric cell division, unveiling this perplexing phenomenon yet a step further.
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